E-selectin antagonist compounds, compositions, and methods of use

ABSTRACT

Methods and compositions using E-selectin antagonists are provided for the treatment and prevention of diseases and disorders treatable by inhibiting binding of E-selectin to an E-selectin ligand. Described herein are E-selectin antagonists including, for example, glycomimetic compounds, antibodies, aptamers and peptides that are useful in methods for treatment of cancers, and treatment and prevention of metastasis, inhibiting infiltration of the cancer cells into bone marrow, reducing or inhibiting adhesion of the cancer cells to endothelial cells including cells in bone marrow, and inhibiting thrombus formation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 61/579,646 filed Dec. 22, 2011, U.S.Provisional Application No. 61/583,547 filed Jan. 5, 2012, U.S.Provisional Application No. 61/704,399 filed Sep. 21, 2012, and U.S.Provisional Application No. 61/704,424 filed Sep. 21, 2012, whichapplications are incorporated by reference herein in their entirety.

BACKGROUND Technical Field

Agents and compositions thereof are described herein that are E-selectinantagonists and may be used as therapeutics. Methods and uses for theseE-selectin antagonists for treating and preventing diseases, disorders,and conditions associated with E-selectin activity are described herein.

Description of the Related Art

Many pathological conditions such as autoimmune and inflammatorydiseases, shock, and reperfusion injuries involve abnormal adhesion ofwhite blood cells. When abnormal adhesion of selectin-mediated celladhesion occurs tissue damage may result instead of repair. Selectinsinclude three cell adhesion molecules that have well-characterized rolesin leukocyte homing. E-selectin (endothelial selectin) and P-selectin(platelet selectin) are expressed by endothelial cells at sites ofinflammation or injury. Recent investigations have suggested that cancercells are immunostimulatory and interact with selectins to extravasateand metastasize (see, e.g., Gout et al., Clin. Exp. Metastasis25:335-344 (2008); Kannagi et al., Cancer Sci. 95:377-84 (2004); Witz,Immunol. Lett. 104:89-93 (2006); Brodt et al., Int. J. Cancer71:612-19(1997)).

A number of cancers are highly treatable when treated before the cancerhas moved beyond the primary site. However, often once the cancer hasspread beyond the primary site, the treatment options are limited andthe survival statistics decline dramatically. For example, whencolorectal cancer is detected at a local stage (i.e., confined to thecolon or rectum), over 90% of those diagnosed survive more than fiveyears. Conversely, when colorectal cancer has spread to distant sites(i.e., metastasized from the primary site to distant sites), thefive-year survival rate of those diagnosed drops dramatically to only11%.

The most common types of cancer include prostate, breast, lung,colorectal, melanoma, bladder, non-Hodgkin lymphoma, kidney, thyroid,leukemias, endometrial, and pancreatic cancers based on estimatedincidence for 2012. The cancer with the highest expected incidence isprostate cancer, with more than 240,000 new cases expected in the U.S.in 2012, and the lowest expected incidence is pancreatic cancer, withapproximately 44,000 new cases expected in 2012.

The highest mortality rate is for patients who have lung cancer. Morethan 160,000 patients are expected to succumb to the disease in 2012.Despite enormous investments of financial and human resources, cancersuch as colorectal cancer remains one of the major causes of death.Colorectal cancer is the second leading cause of cancers-related deathsin the United States of cancers that affect both men and women. Over thelast several years, more than 50,000 patients with colorectal cancerhave died every year.

The four hematological cancers that are most common are acutelymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronicmyelogenous leukemia (CML), and acute myelogenous leukemia (AML).Leukemias and other cancers of the blood, bone marrow, and lymphaticsystem, affect 10 times, more adults than children; however, leukemia isthe most common childhood cancer, and 75% of childhood leukemias areALL. AML is the most common leukemia in adults. Approximately 47,000 newcases are diagnosed every year, and approximately 23,500 people dieevery year from leukemia.

Cancer therapeutic drugs may contribute to endothelial injury, which canin turn cause venous thromboembolism (VTE). Other risk factors thatpredispose an individual to VTE include stasis or endothelial injury(e.g., resulting from indwelling venous device; major trauma or injury),medical conditions, (e.g., malignancy, pregnancy, cardiovascularconditions or events), administration of other drugs such as hormones,and thrombophilia. Blockage of the flow of blood in a body deprivestissue of oxygen and results in damage, destruction or death of thetissue. A thrombus and an embolism can lodge in a blood vessel and blockthe flow of blood. In the United States, approximately 900,000 cases ofVTE, which includes deep venous thrombosis (DVT) and pulmonary embolism(PE), are diagnosed annually and about 300,000 cases are fatal (Heit etal., Blood 2005; 106 (abstract)). Venous thrombosis occurs when redblood cells and fibrin, and to a minor degree, platelets and leukocytesform a mass within an intact vein. Typically, a pulmonary embolismoccurs when a thrombus or a portion of the thrombus detaches from a veinwall and lodges within a pulmonary artery. Because signs and symptoms ofVTE are nonspecific and difficult to diagnose, the exact incidence ofVTE is unknown but may have an annual incidence of 0.1-0.2% (see, e.g.,Anderson, et al., Arch. Intern. Med. 151:933-38 (1991); Silverstein etal., Arch. Intern. Med. 158:585-93 (1998)).

BRIEF SUMMARY

Briefly, provided herein are agents that are E-selectin antagonists,compositions comprising the agents, and methods for using the agents.These agents are useful for treating and preventing diseases anddisorders treatable by inhibiting binding of an E-selectin to anE-selectin ligand, such as cancer, metastasis, and thrombosis amongothers described herein. In certain embodiments, glycomimetic compoundsthat are E-selectin antagonists are provided. Disclosed herein are thefollowing embodiments.

In one embodiment, provided herein is a compound (which is aglycomimetic compound) having the following formula (I):

or a pharmaceutically acceptable salt, isomer, tautomer, hydrate, orsolvate thereof, wherein each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ havethe definitions described herein.

In certain embodiments, R¹ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,C₁-C₈ haloalkyl, C₂-C₈ haloalkenyl or C₂-C₈ haloalkynyl;

-   -   R² is H, or a non-glycomimetic moiety or a        linker-non-glycomimetic moiety, wherein the non-glycomimetic        moiety is selected from polyethylene glycol, thiazolyl,        chromenyl, —C(═O)NH(CH₂)₁₋₄NH₂, C₁-C₈ alkyl, and —C(═O)OY where        Y is C₁-C₄ alkyl, C₂-C₄ alkenyl or C₂-C₄ alkynyl;    -   R³ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈        haloalkyl, C₂-C₈ haloalkenyl, C₂-C₈ haloalkynyl or cyclopropyl;    -   R⁴ is —OH or —NZ¹Z² where Z¹ and Z² are each independently H,        C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl,        C₂-C₈ haloalkenyl or C₂-C₈ haloalkynyl or wherein Z¹ and Z² join        to form a ring;    -   R⁵ is C₃-C₈ cycloalkyl;    -   R⁶ is —OH, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈        haloalkyl, C₂-C₈ haloalkenyl or C₂-C₈ haloalkynyl;    -   R⁷ is —CH₂OH, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈        haloalkyl, C₂-C₈ haloalkenyl or C₂-C₈ haloalkynyl; and    -   R⁸ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl        C₂-C₈ haloalkenyl or C₂-C₈ haloalkynyl.

Additional substructures including, for example compounds of formula(Ia) and other substructures and specific structures of the glycomimeticcompound of formula (I) are described in greater detail herein.Pharmaceutical compositions are also provided that comprise any one ormore of the compounds described above and herein and a pharmaceuticallyacceptable excipient.

Provided herein is a method for treating or preventing metastasis ofcancer cells in a subject, comprising administering to the subject acompound having a structure of formula (I), substructure (Ia), or anyother substructure or specific structure described herein, oradministering a pharmaceutical composition comprising the compound and apharmaceutically acceptable excipient.

In another embodiment, a method is provided for treating or preventingmetastasis of cancer cells in a subject, comprising administering to thesubject a pharmaceutical composition comprising (a), a pharmaceuticallyacceptable excipient, and (b) an agent that is capable of competing witha compound having a structure of formula (I), substructure (Ia), or anyother substructure or specific structure described herein for binding toE-selectin; wherein the agent is an antibody, polypeptide, peptide oraptamer.

In still another embodiment, a method is provided for inhibitinginfiltration of cancer cells into bone marrow in a subject, comprisingadministering to the subject a compound having a structure of formula(I), substructure (Ia), or any other substructure or specific structuredescribed herein, or administering a pharmaceutical compositioncomprising the compound and a pharmaceutically acceptable excipient.

In another embodiment, a method is provided for inhibiting infiltrationof cancer cells into bone marrow in a subject comprising administeringto the subject a pharmaceutical composition comprising (a) apharmaceutically acceptable excipient, and (b) an agent that is capableof competing with a compound having a structure of formula (I),substructure (Ia), or any other substructure or specific structuredescribed herein for binding to E-selectin; wherein the agent is anantibody, polypeptide, peptide or aptamer.

In one embodiment, a method is provided for inhibiting adhesion of atumor cell that expresses a ligand of E-selectin to an endothelial cellexpressing E-selectin, wherein the method comprises contacting theendothelial cell with a compound having a structure of formula (I),substructure (Ia), or any other substructure or specific structuredescribed herein, or administering a pharmaceutical compositioncomprising the compound and a pharmaceutically acceptable excipient,permitting the compound to interact with E-selectin present on theendothelial cell, and thereby inhibiting binding of the tumor cell tothe endothelial cell. In a specific embodiment, the endothelial cell ispresent in the bone marrow.

In another embodiment, a method is provided for treating a cancer in asubject comprising administering to the subject (a) a compound having astructure of formula (I), substructure (Ia), or any other substructureor specific structure described herein, or administering apharmaceutical composition comprising the compound and apharmaceutically acceptable excipient; and (b) at least one of (i)chemotherapy and (ii) radiotherapy.

In still another embodiment, a method is provided for treating orpreventing thrombosis in a subject, comprising administering to thesubject a compound having a structure of formula (I), substructure (Ia),or any other substructure or specific structure described herein, oradministering a pharmaceutical composition comprising the compound and apharmaceutically acceptable excipient.

In yet another a method for treating or preventing thrombosis in asubject, comprising administering to the subject a pharmaceuticalcomposition comprising a pharmaceutical composition comprising (a) apharmaceutically acceptable excipient, and (b) an agent that is capableof competing with a compound having a structure of formula (I),substructure (Ia), or any other substructure or specific structuredescribed herein for binding to E-selectin; wherein the agent is anantibody, polypeptide, peptide or aptamer.

In one embodiment, a method is provided for enhancing hematopoietic stemcell survival in a subject, comprising administering to the subject acompound having a structure of formula (I), substructure (Ia), or anyother substructure or specific structure described herein, oradministering a pharmaceutical composition comprising the compound and apharmaceutically acceptable excipient. In yet another embodiment, amethod is provided for enhancing hematopoietic stem cell survival in asubject, comprising administering to the subject a pharmaceuticalcomposition comprising (a) a pharmaceutically acceptable excipient, and(b) an agent that is capable of competing with a compound having astructure of formula (I), substructure (Ia), or any other substructureor specific structure described herein for binding to E-selectin;wherein the agent is an antibody, polypeptide, peptide or aptamer. Incertain embodiments, the subject has received or will receivechemotherapy or radiotherapy or both chemotherapy and radiotherapy.

Also provided herein is a use of a compound having a structure offormula (I), substructure (Ia), or any other substructure or specificstructure described herein in the manufacture of a medicament fortreating or preventing metastasis of cancer cells.

In another embodiment, provided herein is a use of a compound having astructure of formula (I), substructure (Ia), or any other substructureor specific structure described herein in the manufacture of amedicament for use in combination with chemotherapy or radiotherapy orboth chemotherapy and radiotherapy for treating cancer.

In another embodiment, provided herein is a use of a compound having astructure of formula (I), substructure (Ia), or any other substructureor specific structure described herein in the manufacture of amedicament for treating or preventing thrombosis.

In yet another embodiment, provided herein is a use of a compound havinga structure of formula (I), substructure (Ia), or any other substructureor specific structure described herein in the manufacture of amedicament for inhibiting infiltration of cancer cells into bone marrow.

In still another embodiment, provided herein is a use of a compoundhaving a structure of formula (I), substructure (Ia), or any othersubstructure or specific structure described herein in the manufactureof a medicament for inhibiting adhesion of a tumor cell that expresses aligand of E-selectin to an endothelial cell expressing E-selectin.

In another embodiment, provided herein is a use of a compound having astructure of formula (I), substructure (Ia), or any other substructureor specific structure described herein in the manufacture of amedicament for enhancing hematopoietic stem cell survival.

In another embodiment, a method is provided for treating or preventing(i.e., decreasing or reducing the likelihood of occurrence of)metastasis of cancer cells in an individual (i.e., subject) who is inneed thereof, comprising administering to the individual any one or moreof the glycomimetic compounds of formula (I) described above and hereinor a pharmaceutical composition comprising the compound.

In yet another embodiment, a method is provided for decreasing thelikelihood of occurrence of metastasis of cancer cells in an individualwho is in need thereof, comprising administering to the individual anagent that competes with the compound of formula (I) described above andherein for binding to E-selectin; wherein the agent is an antibody,polypeptide, peptide or aptamer. In certain embodiments, the agent is incombination with a pharmaceutically acceptable excipient (i.e., apharmaceutical composition).

In still another embodiment, a method is provided for decreasing thelikelihood of occurrence of infiltration of cancer cells into bonemarrow in an individual who is in need thereof, said method comprisingadministering to the individual any one or more of the glycomimeticcompounds of formula (I) described above and herein or a pharmaceuticalcomposition comprising the compound.

In another embodiment, a method is provided for decreasing thelikelihood of occurrence of infiltration of cancer cells into bonemarrow in an individual who is in need thereof, comprising administeringto the individual an agent that competes (i.e., is capable of competing)with the compound of formula (I) described above and herein for bindingto E-selectin; wherein the agent is an antibody, polypeptide, peptide oraptamer. In certain embodiments, the agent is in combination with apharmaceutically acceptable excipient (i.e., a pharmaceuticalcomposition).

In yet another embodiment, a method is provided for decreasing thelikelihood of occurrence of thrombus formation in an individual,comprising administering to the individual any one or more of theglycomimetic compounds described above and herein, or a pharmaceuticalcomposition comprising the compounds. In other particular embodiments, amethod is provided for decreasing the likelihood of occurrence ofthrombus formation in an individual, comprising administering to theindividual any one or more of an agent that competes (i.e., is capableof competing) with the compound described above and herein for bindingto E-selectin; wherein the agent is an antibody, polypeptide, peptide oraptamer.

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments.However, one skilled in the art will understand that the invention maybe practiced without these details. In other instances, well-knownstructures have not been shown or described in detail to avoidunnecessarily obscuring descriptions of the embodiments. Unless thecontext requires otherwise, throughout the specification and claimswhich follow, the word “comprise” and variations thereof, such as,“comprises” and “comprising” are to be construed in an open, inclusivesense, that is, as “including, but not limited to.” In addition, theterm “comprising” (and related terms such as “comprise” or “comprises”or “having” or “including”) is not intended to exclude that in othercertain embodiments, for example, an embodiment of any composition ofmatter, composition, method, or process, or the like, described herein,may “consist of” or “consist, essentially of” the described features.Headings provided herein are for convenience only and do not interpretthe scope or meaning of the claimed embodiments.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment. Thus, the appearances of the phrases “in one embodiment” or“in an embodiment” in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments.

Also, as used in this specification and the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “acompound” may refer to one or more compounds, or a plurality of suchcompounds, and reference to “a cell” or “the cell” includes reference toone or more cells and equivalents thereof (e.g., plurality of cells)known to those skilled in the art, and so forth. Similarly, reference to“a composition” includes a plurality of such compositions, and refers toone or more compositions unless the context clearly dictates otherwise.When steps of a method are described or claimed, and the steps aredescribed as occurring in a particular order, the description of a firststep occurring (or being performed) “prior to” (i.e., before) a secondstep has the same meaning if rewritten to state that the second stepoccurs (or is performed) “subsequent” to the first step. The term“about” when referring to a number or a numerical range means that thenumber or numerical range referred to is an approximation withinexperimental variability (or within statistical experimental error), andthus the number or numerical range may vary between 1% and 15% of thestated number or numerical range. It should also be noted that the term“or” is generally employed in its sense including “and/or” unless thecontent clearly dictates otherwise. The term, “at least one,” forexample, when referring to at least one compound or to at least onecomposition, has the same meaning and understanding as the term, “one ormore.”

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (FIG. 1A, FIG. 1B, FIG. 1C and FIG. 1D) is a diagram illustratingthe synthesis of an embodiment (compound 25) of the compounds havingformula I provided herein.

FIG. 2 is a diagram illustrating the synthesis of an embodiment of thecompounds having formula I provided herein.

FIG. 3 is a diagram illustrating that E-selectin plays a central role inthe progression of cancer.

FIG. 4 is a graph depicting the results of a comparison of the effectsof compound 25 (“Cmpd. 25”) of FIG. 1 versus low molecular weightheparin (“LMW heparin”) on the weight of thrombus formed 2 days afterEIM (electrolytic inferior vena cava model) injury. “No treatment”represents the weight of the thrombus 2 days after EIM injury. “Control”(saline) represents venous explant with no injury. “Sham” represents avenous explant 2 days after implantation of the electrode with nocurrent Compound 25 vs. No treatment, P=0.0271; LMW heparin vs. Notreatment, P=0.0203.

FIG. 5 is a graph depicting the results of a comparison of the effectsof compound 25 (“Cmpd. 25”) versus low molecular weight heparin (“LMWH”)on the time required to form a clot.

DETAILED DESCRIPTION

Provided herein are agents that inhibit binding of E-selectin to anE-selectin ligand. The agents include glycomimetic compounds describedherein that inhibit interaction of E-selectin with sialyl Le^(a)(sLe^(a)) or sialyl Le^(x) (sLe^(x)). Agents that are also provided areantibodies, polypeptides, peptides and aptamers that bind at or near thebinding site on E-selectin to which the compounds bind (i.e., anantibody, polypeptide, peptide, or aptamer as described herein that iscapable of competing with the compounds to inhibit E-selectininteraction with sialyl Le^(a) (sLe^(a)) or sialyl Le^(x) (sLe^(x))).

The E-selectin antagonists described herein may be used in methods fortreating a disease or disorder associated with, mediated by, orexacerbated by E-selectin binding to an E-selectin ligand, which in turncauses an undesired biological activity, including, for example, aninflammatory response, promotion of tumor cell migration (i.e.,promoting or enhancing metastasis), enhancing chemotherapy resistance oftumor cells, and contributing to thrombus formation. In certainembodiments, the agents, including the E-selectin antagonistglycomimetic compounds described herein, may be used in the treatment ofcancers in combination with chemotherapy, radiotherapy, or both. Instill other embodiments, the compounds described herein may be used fortreatment and prevention of metastasis of cancer cells (also calledherein tumor cells), including inhibiting infiltration of the cancercells into bone marrow and reducing or inhibiting adhesion of the cancercells to endothelial cells including cells in bone marrow.

Provided herein are agents, such as glycomimetic compounds, thatsignificantly inhibited venous thromboembolism in a treatment model ofthrombus formation and which have certain advantages over currenttreatments of thrombosis. The agents described herein therefore can beused for treating and preventing (i.e., decreasing, inhibiting, orreducing the likelihood of occurrence in a statistical, biological, orclinically significant manner) thrombosis, including deep veinthrombosis and accompanying pulmonary embolism.

E-selectin antagonists (e.g., compounds of formula I) described hereincomprise substituents that are less likely to be cleaved by esterasesand thus have increased stability. These compounds therefore provideimproved compounds than those previously described in the art.

Agents

In one embodiment provided herein, the E-selectin antagonist is aglycomimetic compound that has the following formula (I):

or a pharmaceutically acceptable salt (i.e., physiologically suitablesalt), isomer, tautomer, hydrate or solvate thereof. Formula I comprisesR¹ to R⁸ that represent positions on the compound at which a substituent(e.g., R⁸) or a portion of a substituent (e.g., R³) may be variedaccording to the choices provided herein.

In one embodiment, R¹ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,C₁-C₈ haloalkyl, C₂-C₈ haloalkenyl or C₂-C₈ haloalkynyl;

-   -   R² is H, or a non-glycomimetic moiety or a        linker-non-glycomimetic moiety (i.e., a linker joined to a        non-glycomimetic moiety), wherein the non-glycomimetic moiety is        selected from polyethylene glycol, thiazolyl, chromenyl, C₁-C₈        alkyl, —C(═O)NH(CH₂)₁₋₄NH₂ and —C(═O)OY where Y is C₁-C₄ alkyl,        C₂-C₄ alkenyl or C₂-C₄ alkynyl;    -   R³ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈        haloalkyl, C₂-C₈ haloalkenyl, C₂-C₈ haloalkynyl or cyclopropyl;    -   R⁴ is —OH, or —NZ¹Z², where Z¹ and Z² are each independently H,        C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl,        C₂-C₈ haloalkenyl or C₂-C₈ haloalkynyl or wherein Z¹ and Z² join        to form a ring;    -   R⁵ is C₃-C₈ cycloalkyl;    -   R⁶ is —OH, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈        haloalkyl, C₂-C₈ haloalkenyl or C₂-C₈ haloalkynyl;    -   R⁷ is —CH₂OH, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈        haloalkyl, C₂-C₈ haloalkenyl, or C₂-C₈ haloalkynyl; and    -   R⁸ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈        haloalkyl, C₂-C₈ haloalkenyl or C₂-C₈ haloalkynyl.

In some embodiments, the compound of formula (I) is selected fromcompounds wherein (a) at least one of R¹, R³, R⁶, R⁷ and R⁸ is C₁-C₈haloalkyl; (b) at least one of R³, R⁶, R⁷ and R⁸ is C₁-C₈ haloalkyl; (c)at least two of R¹, R³, R⁶, R⁷ and R⁸ are C₁-C₈ haloalkyl; (d) R² is alinker-non-glycomimetic moiety; or (e) at least one of R¹, R³, R⁶, R⁷and R⁸ is C₁-C₈ haloalkyl, and R² is a linker-non-glycomimetic moiety.

In a particular embodiment of the compound of formula I, C₁-C₈ haloalkylis selected from —CH₂X, —CH₂—(CH₂)_(m)—CH₂X, —CHX₂, —CH₂—(CH₂)_(m)—CHX₂,—CX₃ and —CH₂—(CH₂)_(m)—CX₃, wherein m is 0-6 and X is F, Cl, Br or I.In this embodiment, the terminal carbon is substituted with one or morehalo radicals. In specific embodiments, X is F. When two or more haloradicals are present, each is independently selected. The number ofmethylene groups represented by “m” is “0-6” which includes 0, 1, 2, 3,4, 5, 6 and all ranges between and including 0 to 6. In certainembodiments, at least one of C₁-C₈ haloalkyl is CH₂X, —CHX₂, or —CX₃; incertain more specific embodiments, X is F.

In one embodiment of the compound of formula (I), R¹ is C₁-C₈ alkyl,C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl, C₂-C₈ haloalkenyl forC₂-C₈ haloalkynyl. In certain embodiments of the compound of formula I,R¹ C₁-C₈ alkyl or C₁-C₈ haloalkyl. In more particular embodiments, R¹ isC₁-C₃ alkyl or C₁-C₃ haloalkyl. In a more specific embodiment, R¹ ismethyl (—CH₃), ethyl (CH₂CH₃), or —CF₃ or —CHF₂. In another embodiment,R¹ is methyl (—CH₃) or —CHF₂.

In one embodiment of the compound of formula (I), R² is H, or anon-glycomimetic moiety (M) or a linker (L)-non-glycomimetic moiety,wherein the non-glycomimetic moiety is selected from C₁-C₃ alkyl,—C(═O)NH(CH₂)₁₋₄NH₂, polyethylene glycol (PEG), thiazolyl, chromenyl and—C(═O)OY wherein Y is C₁-C₄ alkyl, C₂-C₄ alkenyl or C₂-C₄ alkynyl. Inone particular embodiment, R² is a non-glycomimetic moiety (M), linker(L)-non-glycomimetic moiety (also indicated as -L-non-glycomimeticmoiety or -L-M), wherein the non-glycomimetic moiety is polyethyleneglycol. In a particular embodiment, R² is —C(═O)NH(CH₂)₂NH₂. In certainembodiments, when R² comprises the non-glycomimetic moiety or alinker-non-glycomimetic moiety described herein, these moieties provideadvantageous or improved characteristics such as enhancedbioavailability; desired pharmacokinetics; improved stability, and thelike, to the compound and are non-immunogenic. Other exemplarynon-glycomimetic moieties described herein include thiazolyl andchromenyl heteroaryls, for example 4-methylthiazolyl and7-hydroxy-2H-chromen-2-on-yl. In some embodiments, R² is H.

R² may be attached to the glycomimetic portion of the compounds offormula (I) either directly or via a linker (L). Linkers are well knownto a person of ordinary skill in the art. In particular embodiments, thelinker that joins the glycomimetic moiety of formula I to anon-glycomimetic moiety (M) is —C(═O)NH(CH₂)₁₋₄NHC(═O)—; in morespecific embodiments, the linker is —C(═O)NH(CH₂)NHC(═O)—, or the linkeris —C(═O)NH(CH₂)₂NHC(═O)—. In other certain embodiments, the linker is—C(═O)NH(CH₂)₁₋₄NHC(═O)(CH₂)₁₋₄; in more specific embodiments, thelinker is —C(O)NH(CH₂)NHC(═O)—CH₂, or the linker is—C(═O)NH(CH₂)₂NHC(═O)—(CH₂)₂. Linkers also include those called in theart “click chemistry” linkers (see, e.g., Brik et al., Chem. Bio. Chem.2003, 4, 1246; Helms et al., J. Am. Chem. Soc. 2004, 126, 15020; Loberet al., Org. Lett. 2003, 5, 1753; Moses et al., Chem. Soc. Rev 2007, 36,1249-1262).

Other exemplary linkers are described in International ApplicationPublication WO 2007/028050. By way of additional example, linkersinclude the following.

In still other embodiments, the linker is

In another embodiment, the linker is —C(═O)—NH—(CH₂)₂—NH—; —CH₂—NH—CH₂—or is —C(═O)—NH—CH₂—.

In one embodiment of the compound of formula (I), R³ is C₁-C₈ alkyl,C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl, C₂-C₈ haloalkenyl, C₂-C₈haloalkynyl or cyclopropyl. In other certain embodiments of the compoundof formula I, R³ is C₁-C₈ alkyl or C₁-C₈ haloalkyl or cyclopropyl. Inmore particular embodiments, R³ is C₁-C₃ alkyl or C₁-C₃ haloalkyl. Inmore specific embodiments, R³ is —CH₃ (methyl) or —CH₂—CH₃ (ethyl) or—CF₃ or —CHF₂. In still other embodiments, R³ is methyl ortrifluoromethyl.

In one embodiment of the compound of formula (I), R⁴ is —OH, or —NZ¹Z²,where —Z¹ and Z² are each independently H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₁-C₈ haloalkyl, C₂-C₈ haloalkenyl or C₂-C₈ haloalkynylor wherein Z¹ and Z² join to form a ring. When Z¹ and Z² join to form aring, the ring is a heterocyclic ring wherein the heteroatom is N. Inone specific embodiment, R⁴ is —OH or —NZ¹Z² wherein Z¹ and Z² are eachH or C₁-C₈ alkyl. In a more specific embodiment, Z¹ and Z² are each —CH₃and —NZ¹Z² is —N(CH₃)₂.

In one embodiment of the compound of formula (I), R⁵ is C₃-C₈ cycloalkyl(i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl orcyclooctyl). In another embodiment, R⁵ is C₃-C₆ cycloalkyl (i.e.,cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl). In a particularembodiment of the compound of formula I, R⁵ is cyclohexyl.

In one embodiment of the compound of formula (I), R⁶ is —OH, C₁-C₈alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl, C₂-C₈ haloalkenylor C₂-C₈haloalkynyl. In other particular embodiments of the compound offormula I, R⁶ is —OH.

In one embodiment of the compound of formula (I), R⁷ is —CH₂OH, C₁-C₈alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl, C₂-C₈ haloalkenylor C₂-C₈ haloalkynyl. In yet another specific embodiment of the compoundof formula I, R⁷ is —CH₂OH, C₁-C₈ alkyl, or C₁-C₈ haloalkyl. In moreparticular embodiments, R⁷ is —CH₂OH or —CH₃. In another specificembodiment, R⁷ is C₁-C₃ haloalkyl. In a more specific embodiment, R⁷ is—CH₂F, —CHF₂ or —CF₃. In another specific embodiment, R⁷ is —CH₂OH or—CHF₂.

In one embodiment of the compound of formula (I), R⁸ is C₁-C₈ alkyl,C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl, C₂-C₈ haloalkenyl orC₂-C₈ haloalkynyl. In another particular embodiment of the compound offormula I, R⁸ is C₁-C₈ alkyl or C₁-C₈ haloalkyl. In more particularembodiments, R⁸ is C₁-C₃ alkyl or C₁-C₃ haloalkyl. In a more particularembodiment, R⁸ is methyl (—CH₃), —CH₂F, —CHF₂ or trifluoromethyl (—CF₃).In another particular embodiment, R⁸ is methyl or trifluoromethyl(—CF₃).

In a particular embodiment of the compound of formula I, at least one orat least two of R¹, R³, R⁶, R⁷ and R⁸ is C₁-C₈ haloalkyl. In othercertain embodiments, at least one of R³, R⁶, R⁷ and R⁸ is C₁-C₈haloalkyl. In other particular embodiments, R² is a linker(L)-non-glycomimetic moiety (M); in still other particular embodiments,R² is a linker (L)-non-glycomimetic moiety (M) and at least one of R¹,R³, R⁶, R⁷ and R⁸ is C₁-C₈ haloalkyl. When two or more of R¹, R³, R⁶, R⁷and R⁸ are C₁-C₈ haloalkyl, the haloalkyls are independently selected,i.e., may be the same or different or both (if at least three present).Oral bioavailability of a compound may be improved and/or the half-lifeof the compound increased when at least one or more of R¹, R³, R⁶, R⁷and R⁸ is C₁-C₈ haloalkyl and when R² comprises a non-glycomimeticmoiety (M) or linker (L)-non-glycomimetic moiety (-L-M).

In another embodiment of the compound of formula (I) provided herein, R⁵is cyclohexyl and R⁶ is —OH and the compound has the following formula(Ia):

or a pharmaceutically acceptable salt (i.e., physiologically suitablesalt), isomer, tautomer, hydrate or solvate thereof,

-   -   wherein R¹ is C₁-C₈ alkyl or C₁-C₈ haloalkyl;    -   R² is H, a non-glycomimetic moiety or a linker-non-glycomimetic        moiety, wherein the non-glycomimetic moiety is selected from        polyethylene glycol, thiazolyl, chromenyl, C₁-C₈ alkyl,        —C(═O)NH(CH₂)₁₋₄NH₂ and —C(═O)OY where Y is C₁-C₄ alkyl;    -   R³ is C₁-C₈ alkyl, C₁-C₈ haloalkyl, or cyclopropyl;    -   R⁴ is —OH or —NZ¹Z² where Z¹ and Z² are each independently H or        C₁-C₈ alkyl;    -   R⁷ is —CH₂OH, C₁-C₈ alkyl, C₁-C₈ haloalkyl, and    -   R⁸ is C₁-C₈ alkyl or C₁-C₈ haloalkyl.

In certain embodiments, halo is F. In other particular embodiments, R¹is —CH₃, —CH₂CH₃, —CH₂F, —CHF₂, —CF₃, —CH₂CH₂F, —CH₂CHF₂, or —CH₂CF₃. Inother embodiments, R³ is —CH₃, —CH₂F, —CHF₂, or —CF₃. In yet anotherparticular embodiment, R⁴ is —OH or —N(CH₃)₂. In certain embodiments, R⁷is —CH₂OH, —CH₃, —CH₂F, —CHF₂, or —CF₃. In still another specificembodiment, R⁸ is —CH₃, —CH₂F, —CHF₂, or —CF₃.

In certain particular embodiments, exemplary compounds of formula (I)are provided, wherein R¹ is ethyl, CF₃, or —CHF₂; R³ is methyl or —CF₃;R⁴ is —OH, or —N(CH₃)₂; R⁵ is cyclohexyl; R⁵ is —OH; R⁷ is —CH₂—OH;—CHF₂, or CF₃; R⁸ is methyl, —CF₃, or —CHF₂; and R² is H, or anon-glycomimetic moiety or linker-non-glycomimetic moiety as describedabove for a compound of formula I. Examples described herein have one ofthe following structural formulae.

In certain particular embodiments, R² is H, —C(═O)NH(CH₂)₂NH₂, or—C(═O)OCH₃ (also depicted as —COOCH₃) and exemplary compounds have oneof the following formulae.

Also provided herein is the following compound of formula (I):

In a particular embodiment of the compound of formula I and formula Ia,R² is a non-glycomimetic moiety that is a polyethylene gylcol (PEG). PEGis a polymer of repeating ethylene oxide units. Length and thusmolecular weight vary depending upon how many of repeating units arepresent. The ethylene oxide units are abbreviated herein as

where n is an integer or a general range of integers from 1 to 100, andany smaller range within the general range. For example the range ofintegers for n may be 1 to 25, 1 to 50, 2 to 15, 2 to 20, 2 to 25, 2 to40, 2 to 50, 2 to 100, 5 to 20, 5 to 40, 5 to 100, as well as all theother numerical combinations. In particular embodiments, n is 4, 8, 12,16, 20, 24, or 28.

In a particular embodiment, PEG is the non-glycomimetic moiety (M) andthe linker (L) is —C(═O)NH(CH₂)₂NHC(═O)— to provide one of the followingcompounds:

wherein n is 1 to 100. In particular embodiments, n is 4, 8, 12, 16, 20,24, or 28.

In two particular embodiments with PEG as R², the compound of formula Ihas one of the following formulae:

In a particular embodiment, R² is a linker-non-glycomimetic moiety, andthe non-glycomimetic moiety is thiazolyl or chromenyl, for example,4-methylthiazolyl or 7-hydroxy-2H-chromen-2-on-yl and the compound offormula (I) has one of the following formulae:

Compounds of formula I include all isomers, physiologically acceptablesalts (i.e., pharmaceutically acceptable salts), hydrates, solvates,polymorphs, metabolites and prodrugs of any. Examples of isomers arestereoisomers (e.g., enantiomers and racemates) and tautomers.

Also provided herein are pharmaceutical compositions that comprise oneor more of the compounds of formula (I), substructures and specificstructures thereof, and a pharmaceutically acceptable excipient. Acompound of formula (I) or a pharmaceutical composition comprising thecompound may be used in methods described herein for treating ofpreventing a disease, disorder, or condition that is treatable byinhibiting (i.e., blocking, reducing, preventing) the interactionbetween E-selectin and a ligand of E-selectin. Such diseases anddisorders include an inflammatory response and related inflammation,cancer, undesired migration or movement of a cell through thevasculature (e.g., metastasis of a tumor-cell), and thrombosis, forexample.

The glycomimetic compounds of formula (I) may be used for treating anyone or more of the diseases or conditions described herein of for thepreparation or manufacture of a medicament for use in treating any oneor more of the diseases or conditions described herein. Each of thesemethods and uses are described in greater detail herein.

Definitions

The terms below, as used herein, have the following meanings, unlessindicated otherwise. Certain chemical groups named herein are precededby a shorthand notation indicating the total number of carbon atoms thatare to be found in the indicated chemical group.

As used herein, a “C₁-C₈ alkyl” or “C₁-C₄ alkyl” refers to an alkanesubstituent with one to eight carbon atoms or one to four carbon atoms,respectively, and may be straight chain, branched, or cyclic (e.g.,cycloalkanyl). The term “alkanyl” may also be used herein and has thesame meaning as alkyl. Examples include methyl (“Me”), ethyl, propyl,isopropyl, butyl and t-butyl. A “C₁-C₈ halo alkyl” refers to a C₁-C₈alkanyl substituted with at least one-halogen (halo); When more than onehalogen is present, the halogens present may be the same or different orboth (if at least three present). A “C₂-C₈ alkenyl” or “C₂-C₄ alkenyl”refers to an alkene substituent with two to eight carbon atoms or two tofour carbon atoms, respectively, at least one carbon-carbon double bond,and may be straight chain, branched or cyclic (cycloalkenyl). Examplesare similar to “C₁-C₈ alkyl” and “C₁-C₈ alkyl” examples except thealkenyl has at least one carbon-carbon double bond. A “C₂-C₈haloalkenyl” refers to a C₂-C₈ alkenyl substituted with at least onehalogen (halo). When more than one halogen is present, the halogenspresent may be the same or different or both (if at least threepresent). A “C₂-C₈ alkynyl” or “C₂-C₄ alkynyl” refers to an alkynesubstituent with two to eight carbon atoms or two to four carbon atoms,respectively, at least one carbon-carbon triple bond, and may bestraight chain, branched, or cyclic (e.g., cycloalkynyl). Examples aresimilar to “C₁-C₈ alkyl” and “C₁-C₈ alkyl” examples except the alkanylhas at least one carbon-carbon triple bond. A “C₁-C₈ haloalkynyl” refersto a “C₂-C₈ alkynyl” substituted with at least one halogen (halo). Whenmore than one halogen is present, the halogens present may be the sameor different or both (if at least three present).

A non-glycomimetic moiety (M) is a moiety that confers one or moreadvantageous properties on the compound that enhance the compound'sefficacy and use in vivo. Examples of such a property include increasedwater solubility, decreased immunogenicity, improved stability, andimproved pharmacokinetic profile. An improved pharmacokinetic profileincludes increased serum half-life, reduced clearance and such thatimprove the therapeutic index.

“Halo” (or “halogen” or “halide”) is fluoro (F), chloro (Cl), bromo(Br), or iodo (I) radical.

“Aryl” refers to a radical derived from a hydrocarbon ring systemcomprising hydrogen, 6 to 30 carbon atoms and at least one aromaticring. The aryl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems. Aryl radicals include, but are not limited to, aryl radicalsderived from the hydrocarbon ring systems of aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane,indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, andtriphenylene. Unless stated otherwise specifically in the specification,the term “aryl” or the prefix “ar-” (such as in “aralkyl”) is meant toinclude aryl radicals that are optionally substituted.

“Aralkyl” refers to a radical of the formula —R_(b)—R_(c) where R_(b) isan alkylene chain as defined above and R_(c) is one or more arylradicals as defined above, for example, benzyl, diphenylmethyl, trityland the like. Unless stated otherwise specifically in the specification,an aralkyl group may be optionally substituted.

“Heterocyclyl”, “heterocycle” or “heterocyclic ring” refers to a stable3- to 24-membered non-aromatic ring radical comprising 2 to 23 carbonatoms and from one to 8 heteroatoms selected from the group consistingof nitrogen, oxygen, and sulfur. In certain embodiments, theheterocyclyl radical is a 5-10 membered heterocycle that comprises 3-9carbon atoms and from 1-3 heteroatoms. Unless stated otherwisespecifically in the specification, the heterocyclyl radical may be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems; nitrogen, carbon or sulfuratom(s) in the heterocyclyl radical may be optionally oxidized; thenitrogen atom may be optionally quaternized; and the heterocyclylradical may be partially or fully saturated. Examples of suchheterocyclyl radicals include, but are not limited to, dioxolanyl,thienyl [1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl,1,1-dioxo-thiomorpholinyl, 12-crown-4, 15-crown-5, 18-crown-6,21-crown-7, aza-18-crown-6, diaza-18-crown-6, aza-21-crown-7, anddiaza-21-crown-7. Unless stated otherwise specifically in thespecification, a heterocyclyl group may be optionally substituted.

“Heterocyclylalkyl” refers to a radical of the formula —R_(b)—R_(c)where R_(b) is an alkylene chain as defined above and R_(c) is one ormore heterocyclyl radicals as defined above, for example,tetrahydrofuranyl-methyl, tetrahydropyranyl-methyl and the like. A6-membered heterocyclylalkyl refers to a heterocyclylalkyl, wherein theheterocyclyl moiety has 6 atoms in the ring. Unless stated otherwisespecifically in the specification, a heterocyclalkyl group may beoptionally substituted.

“Heteroaryl” refers to a 5- to 14-membered ring system radicalcomprising hydrogen atoms, one to thirteen carbon atoms, one to sixheteroatoms selected from the group consisting of nitrogen, oxygen, andsulfur, and at least one aromatic ring. In certain embodiments, theheteroaryl radical is a 5-10 membered heteroaryl that comprises 3-9carbon atoms and from 1-3 heteroatoms. For purposes of this invention,the heteroaryl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heteroarylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized. Examples include, but are not limited, to, azepinyl,acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl,benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl,carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl,furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl,isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl,isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl,oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl,1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl,phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl,pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl,quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl,tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl,triazinyl, and thiophenyl (i.e., thienyl). Unless stated otherwisespecifically in the specification, a heteroaryl group may be optionallysubstituted.

“Heteroarylalkyl” refers to a radical of the formula —R_(b)—R_(c) whereR_(b) is an alkylene chain as defined above and R_(c) is one or moreheteroaryl radicals as defined above, for example, furanyl-methyl,pyridyl-methyl and the like. A 6-membered heteroarylalkyl refers to aheteroarylalkyl, wherein the heteroaryl moiety has 6 atoms in the ring.Unless stated otherwise specifically in the specification, aheteroarylalkyl group may be optionally substituted.

The compounds described herein may generally be used as the free acid orfree base. Alternatively, the compounds may be used in the form of acidor base addition salts. Acid addition salts of the free base aminocompounds may be prepared according to methods well known in the art,and may be formed from organic and inorganic acids. Suitable organicacids include (but are not limited to) maleic, fumaric, benzoic,ascorbic, succinic, methanesulfonic, acetic, oxalic, propionic,tartaric, salicylic, citric, gluconic, lactic, mandelic, cinnamic,aspartic, stearic, palmitic, glycolic, glutamic, and benzenesulfonicacids. Suitable inorganic acids include (but are not limited to)hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acids. Baseaddition salts of the free acid compounds of the compounds describedherein may also be prepared by methods well known in the art, and may beformed from organic and inorganic bases. Suitable inorganic basesincluded (but are not limited to) the hydroxide or other salt of sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum, and the like, and organic bases such as substitutedammonium salts. Thus, the term “pharmaceutically acceptable salt” (orphysiologically suitable salt) of compounds of formula I andsubstructures thereof, as well as any and all substructures and specificcompounds described herein is intended to encompass any and allpharmaceutically suitable salt forms.

Compounds of formula I and substructures thereof and specific structuresmay sometimes be depicted as an anionic species. One of ordinary skillin the art will recognize that the compounds exist with an equimolarratio of cation. For instance, the compounds described herein can existin the fully protonated form, or in the form of a salt such as sodium,potassium, ammonium or in combination with any inorganic base asdescribed above. When more than one anionic species is depicted, eachanionic species may independently exist as either the protonated speciesor as the salt species. In some specific embodiments, the compoundsdescribed herein exist as the sodium salt.

Furthermore, some of the crystalline forms of any compound describedherein may exist as polymorphs, which are also included and contemplatedby the present disclosure. In addition, some of the compounds may formsolvates with water or other solvents. Such solvates are similarlyincluded within the scope of compounds and compositions describedherein.

With regard to stereoisomers, the compounds of formula I as well as anysubstructure or specific structure described herein, may have one ormore chiral (or asymmetric) centers, and may thus give rise toenantiomers, diastereomers, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, as (R)- or (S)-. When thecompounds described herein contain olefinic double bonds or othercenters of geometric asymmetry, and unless specified otherwise, it isintended that the compounds include both E and Z geometric isomers(e.g., cis or trans). Likewise, unless otherwise specified, all possibleisomers, as well as their racemic and optically pure forms, and alltautomeric forms are also intended to be included. It is thereforecontemplated that various stereoisomers and mixtures thereof include“enantiomers,” which refers to two stereoisomers whose molecules arenonsuperimposeable mirror images of one another. Thus, the compounds mayoccur in any isomeric form, including racemates, racemic mixtures, andas individual enantiomers or diastereomers. A tautomer refers to aproton shift from one atom of a molecule to another atom of the samemolecule.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound described herein. Thus, the term “prodrug” refers to ametabolic precursor of a compound described herein that ispharmaceutically acceptable. A prodrug may be inactive when administeredto a subject in need thereof, but is converted in vivo to an activecompound as described herein. Prodrugs are typically rapidly transformedin vivo to yield the parent compound described herein, for example, byhydrolysis in blood. The prodrug compound often offers advantages pfsolubility, tissue compatibility or delayed release in a mammalianorganism (see, e.g., Bundgard, H., Design of Prodrugs (1985), pp. 7-9,21-24 (Elsevier, Amsterdam). A discussion of prodrugs is provided inHiguchi, T., et al., “Pro-drugs as Novel Delivery Systems,” A.C.S.Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design,ed. Edward B. Roche, American Pharmaceutical Association and PergamonPress, 1987, both of which are incorporated in full by reference herein.

The term “prodrug” is also meant to include any covalently bondedcarriers which release the active compound as described herein in vivowhen such prodrug is administered to a mammalian subject. Prodrugs of acompound described herein may be prepared by modifying functional groupspresent in the compound described herein in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent compound described herein. Prodrugs include compoundsdescribed herein wherein a hydroxy, amino or mercapto group is bonded toany group that, when the prodrug of the compound is administered to amammalian subject, cleaves to form a free hydroxy, free amino or freemercapto group, respectively. Examples of prodrugs include, but are notlimited to, ester and amide derivatives of hydroxy, carboxy, mercapto oramino functional groups in the compounds described herein and the like.

Compound Synthesis Procedures

Synthesis of the compounds of formula I (and substructures, and specificcompounds) may be performed as described herein, including the Examples,using techniques familiar to a person skilled in the art. Syntheticmethods for preparing exemplary compounds described herein are describedin Example 1. The methods may be used for synthesis of the compounds offormula I by using appropriate reactants for preparation of the specificcompound using the techniques and methods described herein, and that areroutinely practiced in the art. By way of further example, FIGS. 1 and 2provide schematics of synthesis schemes for exemplary compoundsdescribed herein.

In general, compounds of formula (I) can be prepared according to thefollowing General Reaction Scheme 1:

Referring to General Reaction Scheme 1, compounds of structure A,wherein R¹ and R² are as defined for formula (I), or are moieties whichcan be synthetically converted to R¹ or R², and P¹ is a suitableprotecting group, can be purchased from commercial sources or preparedaccording to methods known in the art. Similarly, compounds of structureB, wherein R⁸ is as defined for formula (I), or is a moiety which can besynthetically converted to R⁸, and P² is a suitable protecting group,can be purchased from commercial sources or prepared according tomethods known in the art. Reaction of A with B, under appropriateconditions (e.g., bromine followed by tetraethylamonium bromide) andsubsequent selective removal of P¹ yields compounds of structure C.

In a parallel scheme, compound D, wherein P³ is a suitable protectinggroup and P⁴ is suitable protecting group or a moiety which can besynthetically manipulated to obtain R³ (as defined for formula (I)), canbe purchased or prepared according to known techniques. Reaction of Dwith a suitable activating agent (e.g., Cl₃CCN) yields activatedcompound E. Other suitable means for activating compounds of structure Dare known to those of ordinary skill in the art. Coupling of C and Eunder appropriate conditions yields compounds of structure F.

Selective removal of P³, followed by selective protection yieldscompounds of structure G, therein P³ is suitable protecting group.Reaction of G with H, wherein P⁶ is suitable protecting group or amoiety which can be synthetically manipulated to obtain R⁴ (as definedfor formula (I)), R⁵ is as defined for formula (I) and LG is a suitablyactivated leaving group (e.g., triflate and the like), and deprotectionyields exemplary compounds of formula (I).

It will be appreciated that further synthetic manipulation may bedesired to obtain certain compounds of formula (I). For example, incertain embodiments, P⁴ may be an allyloxy group which can betransformed to obtain an alkyl amide (e.g., methyl). In other examples,R¹ in the above scheme may be an alkenyl moiety, and the syntheticscheme includes reduction of the alkene to an alkyl group. Various othermodifications to the above General Reaction Scheme I, such as varyingthe starting(s) material or modifying any of the reaction products toinclude other non-hydroxyl moieties at R⁶ and/or R⁷ are possible.Methods for these and other modifications to the above exemplary schemeare well known in the art and described in more detailed in theExamples.

It will also be appreciated by those skilled in the art that in theprocesses described herein the functional groups of intermediatecompounds may need to be protected by suitable protecting groups, evenif not specifically described. Such functional groups include hydroxy,amino, mercapto and carboxylic acid. Suitable protecting groups forhydroxy include trialkylsilyl or diarylalkylsilyl (for example,t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl),tetrahydropyranyl, benzyl, and the like. Suitable protecting groups foramino, amidino and guanidino include t-butoxycarbonyl,benzyloxycarbonyl, and the like. Suitable protecting groups for mercaptoinclude —C(O)—R″ (where R″ is alkyl, aryl or arylalkyl),p-methoxybenzyl, trityl and the like. Suitable protecting groups forcarboxylic acid include alkyl, aryl or arylalkyl esters. Protectinggroups may be added or removed in accordance with, standard techniques,which are known to one skilled in the art and as described herein. Theuse of protecting groups is described in detail in Green, T. W. and P.G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed.,Wiley. As one of skill in the art would appreciate, the protecting groupmay also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

Analogous reactants to those described above may be identified throughthe indices of known chemicals prepared by the Chemical Abstract Serviceof the American Chemical Society, which are available in most public anduniversity libraries, as well as through on-line databases (the AmericanChemical Society, Washington, D.C., may be contacted for more details).Chemicals that are known but not commercially available in catalogs maybe prepared by custom chemical synthesis houses, where many of thestandard chemical supply houses (e.g., those listed above) providecustom synthesis services. A reference for the preparation and selectionof pharmaceutical salts of the present disclosure is P. H. Stahl & C. G.Wermuth “Handbook of Pharmaceutical Salts,” Verlag Helvetica ChimicaActa, Zurich, 2002.

In general, the compounds used in the reactions described herein may bemade according to General Reaction Scheme I, Examples 1 and 2, FIGS. 1and 2 and/or organic synthesis techniques known to those of ordinaryskill in this art, starting from commercially available chemicals and/orfrom compounds described in the chemical literature. “Commerciallyavailable chemicals” may be obtained from standard commercial sourcesincluding Acros Organics (Pittsburgh Pa.), Aldrich Chemical (MilwaukeeWis., including Sigma Chemical and Fluka), Apin Chemicals Ltd. (MiltonPark UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto,Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester Pa.),Crescent Chemical Co. (Hauppauge N.Y.), Eastman Organic Chemicals,Eastman Kodak Company (Rochester N.Y.), Fisher Scientific Co.(Pittsburgh Pa.), Fisons Chemicals (Leicestershire UK), FrontierScientific (Logan Utah), ICN Biomedicals, Inc. (Costa Mesa Calif.), KeyOrganics (Cornwall U.K.), Lancaster Synthesis (Windham N.H.), MaybridgeChemical Co. Ltd. (Cornwall U.K.), Parish Chemical Co. (Orem Utah),Pfaltz & Bauer, Inc. (Waterbury Conn.), Polyorganix (Houston Tex.),Pierce Chemical Co, (Rockford Ill.), Riedel de Haen AG (Hanover,Germany), Spectrum Quality Product, Inc. (New Brunswick, N.J.); TCIAmerica (Portland Oreg.), Trans World Chemicals, Inc. (Rockville Md.),and Wako Chemicals USA, Inc. (Richmond Va.).

Methods known to one of ordinary skill in the art may be identifiedthrough various reference books, articles and databases. Suitablereference books and treatise that detail the synthesis of reactantsuseful in the preparation of compounds of the present disclosure, orprovide references to articles that describe the preparation, includefor example, “Synthetic Organic Chemistry,” John Wiley & Sons, Inc., NewYork; S. R. Sandler et al., “Organic Functional Group Preparations,” 2ndEd., Academic Press, New York, 1983; H. O. House, “Modern SyntheticReactions”, 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L.Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, NewYork, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanismsand Structure,” 4th Ed., Wiley-Interscience, New York, 1992. Additionalsuitable reference books and treatise that detail the synthesis ofreactants useful in the preparation of compounds of the presentdisclosure or provide references to articles that describe thepreparation, include for example, Fuhrhop, J. and Penzlin G. “OrganicSynthesis: Concepts, Methods, Starting Materials”, Second, Revised andEnlarged Edition (1994) John Wiley & Sons ISBN: 3-527-29074-5; Hoffman,R. V. “Organic Chemistry, An Intermediate Text” (1996) Oxford UniversityPress, ISBN 0-19-509618-5; Larock, R. C. “Comprehensive OrganicTransformations: A Guide to Functional Group Preparations” 2nd Edition(1999) Wiley-VCH, ISBN: 0-471-19031-4; March, J. “Advanced OrganicChemistry: Reactions, Mechanisms, and Structure” 4th Edition (1992) JohnWiley & Sons, ISBN: 0-471-60180-2; Otera, J. (editor) “Modern CarbonylChemistry” (2000) Wiley-VCH, ISBN: 3-527-29871-1; Patai, S. “Patai's1992 Guide to the Chemistry of Functional Groups” (1992) InterscienceISBN: 0-471-93022-9; Quin, L. D. et al. “A Guide to OrganophosphorusChemistry” (2000) Wiley-Interscience, ISBN: 0-471-31824-8; Solomons, T.W. G. “Organic Chemistry” 7th Edition (2000) John Wiley & Sons, ISBN:0-471-19095-0; Stowell, J. C., “Intermediate Organic Chemistry” 2ndEdition (1993) Wiley-Interscience, ISBN: 0-471-57456-2; “IndustrialOrganic Chemicals: Starting Materials and Intermediates: An Ullmann'sEncyclopedia” (1999) John Wiley & Sons, ISBN: 3-527-29645-X, in 8volumes; “Organic Reactions” (1942-2000) John Wiley & Sons, in over 55volumes; and “Chemistry of Functional Groups” John Wiley & Sons, in 73volumes.

As noted above, in addition to the compounds described herein, otheragents are provided that bind at or near the binding site on E-selectinfor the compounds and compete with the compounds for the inhibition ofE-selectin interaction with sLe^(a) or sLe^(x). The other agents includeantibodies, polypeptides, peptides and aptamers. Such agents may beproduced by a variety of means that are well known in the art. Forexample, the E-selectin protein is used to generate a library ofantibodies. The library of antibodies is screened for one or moreantibodies of interest using a compound disclosed herein, such ascompound 22 of FIG. 1A. Alternatively, for example, the portion ofE-selectin that binds compound 22 of FIG. 1A is identified and used togenerate antibodies of interest (e.g., use of the portion as animmunogen). This portion of E-selectin may also be used to design andproduce polypeptides, peptides and aptamers that compete with thecompounds described herein.

Antibodies and Antigen-Binding Fragments Thereof

Also provided herein are agents, which may be an antibody, polypeptide,peptide, or aptamer that that are E-selectin antagonists and may beuseful for the methods and uses described herein. Such agents bind toE-selectin at or near the binding site on E-selectin to which a compoundof formula (I) as provided herein binds. These agents are thereforecapable of competing with a compound of formula I to bind to E-selectinand are capable of blocking (i.e., inhibiting) binding of E-selectin toan E-selectin ligand.

An agent includes an antibody, or antigen binding fragment thereof, thatspecifically binds to E-selectin. As described herein, the epitope towhich such an antibody binds comprises amino acids at or near thebinding site on E-selectin to which a compound as provided herein binds.The epitope to which such an antibody binds may include one or moreamino acids contiguous with the residues to which a compound as providedherein binds and/or may include one or more amino acid residues that arenon-contiguous but which interact with the compound.

As used herein, an antibody is said to be “immunospecific,” “specificfor” or to “specifically bind” to an antigen of interest if it reacts ata detectable level with the antigen. Affinities of antibodies andantigen binding fragments thereof can be readily determined usingconventional techniques, for example, those described by Scatchard etal. (Ann. N.Y. Acad. Sci. USA 51:660 (1949)) and by surface plasmonresonance (SPR) (see, e.g., Wolff et al., Cancer Res. b 53:2560 -2565(1993)). Binding properties of an antibody to an antigen may generallybe determined and assessed using immunodetection methods including, forexample, an enzyme-linked immunosorbent assay (ELISA),immunoprecipitation, immunoblotting, countercurrentimmunoelectrophoresis, radioimmunoassays, dot blot assays, inhibition orcompetition assays, and the like, which may be readily performed bythose having ordinary skill in the art (see, e.g., U.S. Pat. Nos.4,376,110 and 4,486,530; Harlow et al., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory (1988)).

These specific antibodies may be polyclonal or monoclonal, prepared byimmunization of animals and subsequent isolation of the antibody, orcloned from specific B cells according to methods and techniquesroutinely practiced in the art and described herein. A variable regionor one or more complementarity determining regions (CDRs) may beidentified and isolated from antigen-binding fragment or peptidelibraries. An antibody, or antigen-binding fragment thereof, may berecombinantly engineered and/or recombinantly produced.

An antibody may belong to any immunoglobulin class. It may be obtainedfrom or derived from an animal, for example, fowl (e.g., chicken) andmammals, which include but are not limited to a mouse, rat, hamster,rabbit, or other rodent, a cow, horse, sheep, goat, camel, human, orother primate. The antibody may be an internalising antibody. Antibodiesmay generally be prepared by any of a variety of techniques known topersons having ordinary skill in the art and described herein. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory (1988); Peterson, ILAR J. 46:314-19 (2005); Kohler andMilstein (Nature 256:495-97 (1976); Eur. J. Immunol 6:511-19 (1975);Coligan et al. (eds.), Current Protocols in Immunology, 1:2.5.1-2.6.7(John Wiley & Sons 1991)).

Human monoclonal anti-E-selectin antibodies may be generated by anymember of techniques with which those having ordinary skill in the artwill be familiar (see, e.g., U.S. Pat. No. 4,464,456; Lonberg et al.,Nature 368:856 (1994); U.S. Pat. No. 5,877,397; Braggemann et al., Curr.Opin. Biotechnol. 8:455-58 (1997); Jakobovits et al., Ann. N. Y. Acad.Sci. 764:525-35 (1995)); (WO 92/02551; U.S. Pat. No. 5,627,052; Babcooket al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996); or otherprocedures as known in the art). Chimeric antibodies, specific for theportion of E-selectin of interest, including humanized chimericantibodies, may also be generated. See, e.g., Morrison et al., Proc.Natl. Acad. Sci. USA, 81:6851-55 (1984); Shin et al., Methods Enzymol.178:459-76 (1989)). Strategies for designing humanized antibodies areroutinely practiced in the art (see, e.g., Jones et al., Nature321:522-25 (1986); Riechmann et al., Nature 332:323-27 (1988); Padlan etal., FASEB 9:133-39 (1995); Chothia et al., Nature, 342:377-83 (1989);Bajorath et al., Ther. Immunol. 2:95-103 (1995)).

For particular uses, antigen-binding fragments of antibodies may bedesired. Antibody fragments, F(ab′)₂, Fab, Fab′, Fv, and Fd, can beobtained, for example, by proteolytic hydrolysis of the antibody (see,e.g., Weir, Handbook of Experimental Immunology, Blackwell Scientific,Boston (1986)), or may be synthetically prepared or geneticallyengineered. Antibody fragments include recombinant single chainpolypeptide molecules in which light and heavy variable regions areconnected by a peptide linker (scFv proteins), and minimal recognitionunits (comprises at least one CDR) consisting of the amino acid residuesthat mimic the hypervariable region. Methods and techniques forpreparing and isolating antibody fragments are described in the art(see, e.g., Larrick et al., Methods: A Companion to Methods inEnzymology 2:106, (1991); Courtenay-Luck, in Monoclonal Antibodies:Production, Engineering and Clinical Application, Ritter et al. (eds.),page 166 (Cambridge University Press 1995); and Ward et al., inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995); International PatentApplication Nos. PCT/US91/08694 and PCT/US91/04666); Scott et al.,Science 249:386 (1990); Devlin et al., Science 249:404 (1990); Cwirla etal., Science 276:1696-99 (1997); U.S. Pat. No. 5,223,409; U.S. Pat. No.5,733,731; U.S. Pat. No. 5,498,530; U.S. Pat. No. 5,432,018; U.S. Pat.No. 5,338,665; U.S. Pat. No. 5,922,545; International ApplicationPublication Nos. WO 96/40987 and WO 98/15833).

Antibodies may also be identified and isolated from human, rabbit, mouseor chicken immunoglobulin phage libraries. Antibodies isolated fromnon-human species or non-human immunoglobulin libraries may begenetically engineered to “humanize” the antibody or fragment thereof.See, e.g., Winter et al., Annu. Rev. Immunol. 12:433-55 (1994); Burtonet al., Adv. Immunol 57:191-280 (1994); U.S. Pat. No. 5,223,409; Huse etal., Science 246:1275-81 (1989); Kang et al., Proc. Natl. Acad. Sci. USA88:4363-66 (1991); Hoogenboom et al., J. Molec. Biol. 227:381-388(1992); U.S. Pat. No. 6,703,015).

An agent that is an E-selectin antagonist also includes apeptide-immunoglobulin (Ig) constant region fusion polypeptide, whichincludes a peptide-IgFc fusion polypeptide. The peptide may be anynaturally occurring or recombinantly prepared molecule. A peptide-Igconstant region fusion polypeptide, such as a peptide-IgFc fusionpolypeptide (also referred to in the art as a peptibody (see, e.g., U.S.Pat. No. 6,660,843)), comprises a biologically active peptide orpolypeptide capable of altering the sLe^(a) or sLe^(x) binding functionof E-selectin that is fused in-frame with a portion, at least oneconstant region domain (e.g., CH1, CH2, CH3, and/or CH4). Antibodyrelated sequences are provided in Kabat et al. (in Sequences of Proteinsof Immunological Interest, 4th ed. (U.S. Dept. of Health and HumanServices, U.S. Government Printing Office, 1991).

Peptides and Peptidomimetics

In certain embodiments, interaction between E-selectin and sLe^(a) orsLe^(x) may be inhibited (i.e., inhibited, decreased, disrupted reducedin a biologically or statistically significant manner) by a peptide orpeptidomimetic of the portion of E-selectin that binds a compoundprovided herein. The peptide and the peptide moiety of thepeptidomimetic may comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, or 46-50 aminoacids, Peptides and peptidomimetics typically have molecular masses lessthan 10⁴ daltons, less than 10³ daltons, or less than 10² daltons.

Methods of Use

Methods are provided herein for using any one or more of the E-selectinantagonist agents described above and herein, including glycomimetics offormula (I), antibodies or antigen-binding fragments thereof,polypeptides, peptides and aptamers for preventing (i.e., reducing thelikelihood of occurrence or recurrence of) and/or treating a disease ordisorder associated with, mediated by, or exacerbated by E-selectinbinding to an E-selectin ligand, which in turn causes an undesiredbiological activity. Thus, the E-selectin antagonists described hereinmay be used in methods for treating a disease or disorder treatable byinhibiting binding of E-selectin to an E-selectin ligand. These methodsand other embodiments are described in greater detail herein.

In certain embodiments, a compound of formula (I) or a pharmaceuticalcomposition comprising the compound may be used in methods for treatingand preventing (i.e., decreasing or reducing the likelihood ofoccurrence of) metastasis of cancer cells (also called tumor cellsherein) in an individual (i.e., subject, patient) who is in need thereofby administering the compounde or composition to the individual. Inother embodiments, a compound of formula (I) or a pharmaceuticalcomposition comprising the compound may be used in methods forinhibiting (reducing, decreasing, or preventing (i.e., decreasing thelikelihood of occurrence of)) infiltration of cancer cells into bonemarrow in an individual (i.e., subject, patient) who is in need thereofby administering the compound or composition to the individual. In stillanother embodiment, methods are provided herein for inhibiting(reducing, decreasing, or preventing) adhesion of a cancer cell thatexpresses a ligand of E-selectin to an endothelial cell expressingE-selectin on the cell surface of the endothelial cell wherein themethod comprises contacting the endothelial cell and the compound orcomposition comprising the compound (i.e., in some manner permitting thecompound or composition comprising the compound to interact with theendothelial cell) such that when the compound interacts with E-selectinon the endothelial cell, binding of the cancer cell to the endothelialcell is inhibited. In certain embodiments, the endothelial cell ispresent in the bone marrow. In other embodiments, an E-selectinantagonist agent selected from an antibody or antigen-binding fragmentthereof, polypeptide, peptide and aptamer, which agent is capable ofcompeting with a compound of formula (I), may be used in theaforementioned methods.

In still another embodiment described herein, a method is providing fortreating a cancer in an individual (i.e., subject, patient) byadministering a compound of formula I or a pharmaceutical compositioncomprising the compound to the individual. The compound (orpharmaceutical composition comprising the compound) may be administeredin conjunction with (i.e., as an adjunct therapy, which is also calledadjunctive therapy) with chemotherapy or radiation or both. Thechemotherapy or radiation therapy or combination may be referred to asthe primary anti-tumor or anti-cancer therapy that is being administeredto the individual to treat the particular cancer. In other embodiments,an E-selectin antagonist agent selected from an antibody orantigen-binding fragment thereof, polypeptide, peptide and aptamer,which agent is capable of competing with a compound of formula (I), maybe used in the aforementioned methods.

In still another embodiment, a compound of formula I or pharmaceuticalcompositions comprising the compound may be used in methods forenhancing hematopoietic stem cell survival in a subject. In otherembodiments, an E-selectin antagonist agent selected from an antibody orantigen-binding fragment thereof, polypeptide, peptide and aptamer,which agent is capable of competing with a compound of formula (I), maybe used in the aforementioned methods.

In another embodiment, a compound of formula I or pharmaceuticalcompositions comprising the compound may be used in methods for treatingor preventing (i.e., decreasing or reducing the likelihood or risk ofoccurrence of) thrombosis in a subject. In certain embodiments, acompound of formula I or pharmaceutical compositions comprising thecompound may be used in methods for treating or preventing (i.e.,decreasing or reducing the risk of occurrence of) thrombus formation inan individual who is need of such treatment, comprising administering tothe individual a compound having the formula (I) (or the pharmaceuticalcomposition comprising the compound), or any substructure or specificstructure described herein. In other embodiments, an E-selectinantagonist agent selected from an antibody or antigen-binding fragmentthereof, polypeptide, peptide and aptamer, which agent is capable ofcompeting with a compound of formula (I), may be used in theaforementioned methods.

As understood by a person of ordinary skill in the medical art, theterms, “treat” and “treatment,” refer to medical management of adisease, disorder, or condition of a subject (i.e., patient, individual)(see, e.g., Stedman's Medical Dictionary). In general, an appropriatedose and treatment regimen provide at least one glycomimetic compound orother agent described herein in an amount sufficient to providetherapeutic and/or prophylactic benefit. Therapeutic and/or prophylacticbenefit includes, for example, an improved clinical outcome, boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow or retard (lessen) an undesiredphysiological change or disorder, or to prevent or slow or retard(lessen) the expansion or severity of such disorder. As discussedherein, beneficial or desired clinical results from treating a subjectinclude, but are not limited to, abatement, lessening, or alleviation ofsymptoms that result from or are associated with the disease, condition,or disorder to be treated; decreased occurrence of symptoms; improvedquality of life; longer disease-free status (i.e., decreasing thelikelihood or the propensity that a subject will present symptoms on thebasis of which a diagnosis of a disease is made); diminishment of extentof disease; stabilized (i.e., not worsening) state of disease; delay ofslowing of disease progression; amelioration or palliation of thedisease state; and remission (whether partial or total), whetherdetectable or undetectable; and/or overall survival. “Treatment” canalso mean prolonging survival when compared to expected survival if asubject were not receiving treatment. Subjects in need of treatmentinclude those who already have the disease, condition, or disorder aswell as subjects prone to have or at risk of developing the disease,condition, or disorder, and those in which the disease, condition, ordisorder is to be prevented (i.e., decreasing the likelihood ofoccurrence of the disease, disorder, or condition).

As discussed in detail herein, the disease or disorder to be treated orprevented (i.e., reduce the likelihood of occurrence or recurrence) is acancer and related metastasis and includes cancers that comprise solidtumor(s) and cancers that comprise liquid tumor(s). As illustrated inFIG. 3, E-selectin plays a central role in the progression of a cancer.The invasive properties of cancer cells depend, at least in part, on thecapability of the cancer cell to breach the endothelial barrier. Cancercells, for example, colon cancer cells, may express E-selectin ligandsthat are capable of binding to endothelial cells that express E-selectinon their cell surface. Without wishing to be bound by theory, binding ofthe cancer cell to the endothelial cell can contribute to extravasationof the cancer cells (see, e.g., Tremblay et al., Oncogene 25: 6563-6573,doi:10.1038/sj.onc.1209664; published online 22 May 2006).

Cancers that may be prevented from metastasizing includes cancers thatcomprise solid tumors and those that comprise liquid tumors (e.g.,hematological malignancies). Examples of solid tumors that may betreated with the agents described herein (e.g., glycomimetic compoundsof formula I) include colorectal cancer, liver cancer, gastric cancer,lung cancer, brain cancer, kidney cancer, bladder cancer, thyroidcancer, prostate cancer, ovarian cancer, cervical cancer, uterinecancer, endometrial cancer, melanoma, breast cancer, and pancreaticcancer. Liquid tumors occur in the blood, bone marrow, and lymph nodesand include leukemia (e.g., AML, ALL, CLL, and CML), lymphoma (e.g.,non-Hodgkin lymphoma and Hodgkin lymphoma), and myeloma (e.g., multiplemyeloma). Reports have described that liquid tumors such as multiplemyeloma follow a similar invasion—metastasis cascade as observed withsolid tumors and that E-selectin ligands are present on liquid tumorcells, such as myeloma cells (see, e.g., Ghobrial, Blood 120:20-30(2012) Epub 2012 Apr. 24). Others have observed that ligands ofE-selectin (e.g., CD65) may be important for extravascular infiltrationof leukemia cells (see, e.g., Noguchi et al., Leukemia Res. 25:847-53(2001)). Liquid tumor cells may also adhere to bone marrow, which mayfurther lead to sequestration and quiescence of the tumor cells,resulting in “resistance” of the tumor cells to chemotherapy, whichphenomenon is referred to as adhesion mediated drug resistance. Studieshave also indicated that bone marrow contains anatomic regions thatcomprise specialized endothelium, which expresses the E-selectin (see,e.g., Sipkins et al., Nature 435:969-973 (2005)). Accordingly, anE-selectin antagonist, such as those described herein, may be useful forinhibiting metastasis of cancers that comprise either a solid or liquidtumor by inhibiting binding of an E-selectin ligand to E-selectin.

In particular embodiments, the compounds of formula (I), includingsubstructures and specific compounds, and agents described herein may beused for treating or preventing (i.e., decreasing or reducing thelikelihood of occurrence of) metastasis of cancer cells in an individual(i.e., subject, patient) who is in need thereof. The compounds andagents described herein may be used for inhibiting or preventing (i.e.,decreasing or reducing the likelihood of occurrence of) infiltration ofcancer cells into bone marrow in an individual who is in need thereof.The individuals (or subjects) in need of such treatments includesubjects who have been diagnosed with a cancer, either a cancer thatcomprises solid tumor(s) or a cancer that comprises a liquid tumor.Without wishing to be bound by theory, by inhibiting tumor cells frommetastasizing to the bone marrow or to other protective niches in thebody, the tumor cells are inhibited from sequestration and protectionfrom exposure to chemotherapy or radiotherapy.

Such cancers included, for example, colorectal cancer, liver cancer,gastric cancer, lung cancer, brain cancer, kidney cancer, bladdercancer, thyroid cancer, prostate cancer, ovarian cancer, cervicalcancer, uterine cancer, endometrial cancer, melanoma, breast cancer, andpancreatic cancer. Liquid tumors occur in the blood, bone marrow, thesoft, sponge-like tissue in the center of most bones, and lymph nodesand include leukemia (e.g., AML, ALL, CLL, and CML), lymphoma, andmyeloma (e.g., multiple myeloma). Lymphomas include Hodgkin lymphoma,which is marked by the presence of a type of cell called theReed-Sternberg cell; and non-Hodgkin lymphomas, which includes a large,diverse group of cancers of immune system cells. Non-Hodgkin lymphomascan be further divided into cancers that have an indolent (slow-growing)course and those that have an aggressive (fast-growing) course, andwhich subtypes respond to treatment differently.

The compounds of formula I and agents described herein (or thepharmaceutical composition comprising the compound or agent) may beadministered as an adjunct therapy to chemotherapy or radiotherapy orboth, which is being delivered to the subject as primary therapy fortreating the cancer. The chemotherapy and radiotherapy that may beadministered depend upon several factors including the type of cancer,location of the tumor(s), stage of the cancer, age and gender andgeneral health status of the subject. A person skilled in the medicalart can readily determine the appropriate chemotherapy regimen orradiotherapy regimen for the subject in need. The person skilled in themedical art can also determine, with the aid of preclinical and clinicalstudies, when the compound of formula (I) or agent should beadministered to the subject, that is whether the compound or agent isadministered prior to, concurrent with, or subsequent to a cycle of theprimary chemotherapy or radiation treatment.

Also provided herein is a method for inhibiting adhesion of a tumor cellthat expresses a ligand of E-selectin to an endothelial cell expressingE-selectin on its cell surface, which method comprises contacting theendothelial cell with the compound of formula (I) or agent as describedherein, thereby permitting the compound to interact with E-selectin onthe endothelial cell surface and inhibiting binding of the tumor cell tothe endothelial cell. Without wishing to be bound by theory, inhibitingadhesion of tumor cells to endothelial cells may reduce in a significantmanner, the capability of the tumor cells to extravasate into otherorgans, blood vessels, lymph, or bone marrow and thereby reduce,decrease, or inhibit, or slow the progression of the cancer, includingreducing, decreasing, inhibiting, of slowing metastasis.

In particular embodiments of the methods described herein, the subjectis a human or non-human animal. A subject in need of the treatmentsdescribed herein may exhibit symptoms or sequelae of cancer disease,disorder, or condition described herein or may be at risk of developingthe disease, disorder, or condition. Non-human animals that may betreated include mammals, for example, non-human primates (e.g., monkey,chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils,hamsters, ferrets, rabbits), lagomorphs; swine (e.g., pig, miniaturepig), equine, canine, feline, bovine, and other domestic, farm, and zooanimals.

The effectiveness of a compound, agent, or pharmaceutical compositiondescribed herein in treating or preventing a disease or disorder orcondition described herein; and determining and adjusting an appropriatedosing regimen (e.g., adjusting the amount of compound per dose and/ornumber of doses and frequency of dosing), can readily be determined by aperson of ordinary skill in the medical and clinical arts. One or anycombination of diagnostic methods, including physical examination,assessment and monitoring of clinical symptoms, and performance ofanalytical tests and methods described herein, may be used formonitoring the health status of the subject.

As described herein, with respect to treating a subject (i.e., anindividual) who has cancer or who is at risk of developing cancer, atleast one (i.e., one or more) of the above described agents (e.g.,compounds of formula (I)) may be administered in combination with atleast one (i.e., one or more) additional anti-cancer agent. Chemotherapymay comprise one or more chemotherapeutic agents. For example,chemotherapy agents, radiotherapeutic agents, inhibitors ofphosphoinositide-3 kinase (PI3K), and inhibitors of VEGF may be used incombination with an agent described herein. Examples of inhibitors ofPI3K include the compound named by Exelixis as “XL499.” Examples of VEGFinhibitors include the compound called “cabo” (previously known asXL184). Many other chemotherapeutics are small organic molecules. Asunderstood by a person skilled in the art, chemotherapy may also referto a combination of two or more chemotherapeutic molecules that areadministered coordinately and which may be referred to as combinationchemotherapy. Numerous chemotherapeutic drugs are used in the oncologyart and include, for example, alkylating agents; antimetabolites;anthracyclines, plant alkaloids; and topoisomerase inhibitors.

An E-selectin antagonist, such as a glycomimetic compound of formula (I)may function independent of the anti-cancer agent, or may function incoordination with the anti-cancer agent, e.g., by enhancingeffectiveness of the anti-cancer agent or vice versa. In one embodiment,methods are provided for treating cancers comprising administering theE-selectin antagonists described herein (including the glycomimeticcompounds of formula I). The cancer may be a solid tumor or a liquidtumor. In certain embodiments, the E-selectin antagonist is used incombination with chemotherapy, radiation, or both chemotherapy andradiation. The E-selectin antagonist may be administered with one ormore cycles (i.e., one, two, three, four, five, six, or more cycles) ofchemotherapy or radiotherapy when multiple cycles of the chemotherapy orradiotherapy are administered to a subject for the treatment of acancer. The E-selectin antagonist may enhance the efficacy of thechemotherapeutic agent(s) or radiotherapy.

In another embodiment, provided herein ate methods for enhancing (i.e.,enhancing, promoting, improving the likelihood of, enhancing in astatistically or biologically significant manner) or maintainingsurvival of hematopoietic stem cells (HSC) in a subject who is treatedwith or will be treated with a chemotherapeutic drug(s) or radioactivetherapy, respectively, comprising administering one or more of theE-selectin antagonist glycomimetic compounds described herein. Incertain embodiments, the subject receives or will receive bothchemotherapy and radiation therapy. Also, provided herein is a methodfor reducing (i.e., reducing, inhibiting, diminishing in a statisticallyor biologically significant manner) chemosensitivity or radiosensitivityof hematopoietic stem cells (HSC) to the chemotherapeutic drug(s) orradioactive therapy, respectively, in a subject. Because repeated cyclesof chemotherapy and radiotherapy often diminish the ability of HSCs torecover and replenish bone marrow, the glycomimetic compounds describedherein may be useful for subjects who will receive more than one cycle,such as at least two, three, four or more cycles, of chemotherapy orradiotherapy or a combination of both chemotherapy and radiotherapy. TheE-selectin antagonist may therefore be administered with any one or moreof each of the cycles of chemotherapy or radiotherapy (or combination)administered to the subject. HSCs reside in the bone marrow and generatethe cells that are needed to replenish the immune system and the blood.Anatomically, bone marrow comprises a vascular niche that is adjacent tobone endothelial sinuses (see, e.g., Kiel et al., Cell 121:1109-21(2005); Sugiyama et al., Immunity 25:977-88 (2006); Mendez-Ferrer etal., Nature 466:829-34 (2010); Butler et al., Cell Stem Cell 6:251-64(2010)). A recent study describes that E-selectin promotes HSCproliferation and is an important component of the vascular niche (see,e.g., Winkler et al., Nature Medicine published online 21 Oct. 2012;doi:10.1038/nm.2969; see also, e.g., Int'l. Patent Appl. Publ. No.2007/028050). Deletion or inhibition of E-selectin enhanced HSC survivalin mice that were treated with chemotherapeutic agents or radiotherapyand accelerated blood neutrophil recovery (see, e.g., Winkler et al.,supra).

An agent described herein (i.e., an E-selectin antagonist, such as aglycomimetic compound of formula (I)) may function independent of theanti-cancer agent, or may function in coordination with the anti-canceragent, e.g., by enhancing effectiveness of the anti-cancer agent or viceversa. In addition, the administration of one or more of the E-selectinantagonist agents described herein may be in conjunction with one ormore other therapies, e.g., for reducing toxicities of therapy. Forexample, at least one (i.e., one or more) palliative agent to counteract(at least in part) a side effect of therapy (e.g., anti-cancer therapy)may be administered. Agents (chemical or biological) that promoterecovery, or counteract side effects of administration of antibiotics,or corticosteroids, are examples of such palliative agents. At least oneagent described herein may be administered before, after, orconcurrently with administration of at least one additional anti-canceragent or at least one palliative agent to reduce a side effect oftherapy. Where administration is concurrent, the combination may beadministered from a single container or two (or more) separatecontainers.

Cancer cells (also called herein tumor cells) that may be prevented(i.e., inhibited, slowed) from metastasizing, may be killed, may beprevented from adhering to an endothelial cell, or inhibited frominfiltrating bone marrow include cells of solid tumors and liquid tumors(including hematological malignancies). Examples of solid tumors aredescribed herein and include colorectal cancer, liver cancer, gastriccancer, lung cancer, brain cancer, kidney cancer, bladder cancer,thyroid cancer, prostate cancer, ovarian cancer, cervical cancer,uterine cancer, endometrial cancer, melanoma, breast cancer, andpancreatic cancer. Liquid tumors occur in the blood, bone marrow, andlymph nodes and include leukemia (e.g., AML, ALL, CLL, and CML),lymphoma (e.g., Hodgkin lymphoma and non-Hodgkin lymphoma), and myeloma(e.g., multiple myeloma). As used herein, the term cancer cells includemature, progenitor and cancer stem cells.

Bones are a common location for cancer to infiltrate once leaving theprimary tumor location. Once cancer resides in bone, it is frequently acause of pain to the individual. In addition, if the particular boneaffected is a source for production of blood cells in the bone marrow,the individual may develop a variety of blood cell related disorders.Breast and prostate cancer are examples of solid tumors that migrate tobones. Acute myelogenous leukemia (AML) and multiple myeloma (MM) areexamples of liquid tumors that migrate to bones. Cancer cells thatmigrate to bone will typically migrate to the endosteal region of thebone marrow. Once cancer cells have infiltrated into the marrow, thecells become quiescent and are protected from chemotherapy. Thecompounds of the present invention block infiltration of disseminatedcancer cells into bone marrow. A variety of individuals may benefit fromtreatment with the compounds. Examples of such individuals includeindividuals with a cancer type having a propensity to migrate to bonewhere the tumor is still localized or the tumor is disseminated but notyet infiltrated bone, or where individuals with such a cancer type arein remission.

The cancer patient population most likely to respond to treatments usingthe agents (e.g., compounds of formula (I)) described herein can beidentified based on the mechanism of action of E-selectin. That is,patients may be selected that express a highly active E-selectin asdetermined by the genetic polymorphism for E-selectin of S128R(Alessandro et al., Int. J. Cancer 121:528-535, 2007). In addition,patients for treatment by the agents described herein may also selectedbased on elevated expression of the E-Select in binding ligands (sialylLe^(a) and sialyl Le^(x)) as determined by antibodies directed againstcancer-associated antigens CA-19-9 (Zheng et al., World J.Gastroenterol. 7:431-434, 2001) and CD65. In addition antibodiesHECA-452, and FH-6 which recognize similar carbohydrate ligands ofE-selectin may also be used in a diagnostic assay to select the cancerpatient population most likely to respond to this treatment.

In other embodiments, methods are provided for treating or preventing(i.e., reducing the likelihood of occurrence) thrombosis in a subject(i.e., individual, patient) in need thereof. The subject may have athrombus or may be at risk of developing a thrombus. An E-selectinantagonist described herein (including a compound of formula (I)) mayinhibit or prevent (i.e., reduce the likelihood of occurrence of) theformation of a thrombus. The E-selectin antagonist may inhibit slow orretard formation of a thrombus or decrease the size or integrity of aformed thrombus. While this method is applicable to individuals in needthereof generally, the methods are especially advantageous for suchindividuals who are also at risk for bleeding. For example, this methodis useful and advantageous in a wide variety of situations in which therisk of bleeding is significant and the use of anti-thrombosis agentswith anti-coagulant properties (such as LMW heparin) is contraindicated.Even when the use of an anti-thrombosis agent with anti-coagulantproperties is not believed to be contraindicated, this method provides abenefit if bleeding nevertheless occurs. The E-selectin antagonists usedin the method are agents that inhibit the interaction of E-selectin withsialyl Le^(a) (sLe^(a)) or sialyl Le^(x) (sLe^(x)), but in contrast toagents, such as heparin, do not significantly delay clotting.

Selectin-mediated activation of leukocytes promotes formation ofprocoagulant microparticles rich in tissue factor (see, e.g., Wakefieldet al., Thrombosis Res. 123:S3, 5-40 (2009)). Both E and P-selectins areexpressed on the endothelium after injury or activation of the bloodvessel wall. Many reports have focused on the role of P-selectin inthrombosis, in part due to the availability of inhibitors for P-selectin(see, e.g., Lopez et al., Hematology Am. Soc. Hematol. Educ. Program439-56 (2004)); however, several studies conclude E-selectin has adominant role. Without wishing to be bound by any particular theory, theformation of VTE is driven by the inflammatory response, and theselectins function in early events of thrombosis.

Various drugs are currently used for the treatment of thrombosis.Exemplary drugs include those that suppress platelet aggregation(anti-platelet therapeutics), for example, aspirin, ticlopidine,eicosapentaenoic acid (EPA), dipyridamole, and dilazep hydrochloride. Ananti-platelet therapeutic such as aspirin suppresses formation ofthrombus at the impaired site of the blood vessel by suppressingdevelopment of blood coagulation triggered by platelet aggregation.However, because platelets also prevent hemorrhage from the bloodvessel, excessive suppression of the platelet can result in decreasedeffectiveness of platelets in preventing hemorrhage.

Anticoagulants used for treatment or prevention of thrombosis act bysuppressing a blood coagulation factor and include warfarin, heparin,low molecular weight heparin, and argatroban. Anticoagulants are usefulin preventing formation of intravascular fibrin clots, whereasfibrinolytics (e.g., plasminogen activators) are useful for dissolutionof fibrin clots. Uncontrolled bleeding may occur after long-termadministration of large doses of an anticoagulant or fibrinolytic. Whenheparin is used, complications include heparin resistance, bleeding,heparin-induced thrombocytopenia, and osteoporosis.

E-selectin, in particular, plays a dominant role in thrombus formation,which was determined in animal models and by studying humans who have agenetic polymorphism (Ser128Arg) of E-selectin. In human studies, asingle nucleotide polymorphism (SNP) S128R (serine to arginine atposition 128) in the E-selectin gene is reported to be overrepresentedin patients with atherosclerosis, restenosis, coronary heart disease,myocardial infarction, and colorectal cancer with poor prognosis (Myerset al., J. Surg. Res. 108:212-21 (2002)). S128R E-selectin is a geneticvariant that is more active than normal (i.e., wild-type) E-selectin.Cells expressing S128R E-selectin show greater adhesion and adhere to awider variety of cell types (Yoshida et al., Arterioscler. Thromb. Vasc.Biol. 23:783-88 (2003)). According to publications and a screen by theConsortium of Functional Glycomics, S128R E-selectin binds to the samecarbohydrates (sialyl Le^(a) and sialyl Le^(x)) as the wild typeE-selectin, although its binding activity in other in vitro assays isenhanced and perhaps more promiscuous.

In a study on the effects of S128R E-selectin on venous thromboembolism(VTE), 585 patients were prospectively observed after the first VTE forrecurrence after discontinuation of treatment (see, e.g., Jilma et al.,Arch. Intern. Med. 166:1655-59 (2006)). Patients with S128R E-selectinshowed a significant increase in developing another thrombus afterstopping anticoagulant therapy when compared to patients with wild typeE-selectin.

In an embodiment, the thrombosis is a venous thromboembolism (VTE). VTEcauses deep vein thrombosis and pulmonary embolism. Low molecular weight(LMW) heparin is the current mainstay therapy for the prevention andtreatment of VTE. There are many circumstances, however, when the use ofLMW heparin is contraindicated. Patients undergoing surgery, patientswith thrombocytopenia, patients with a history of stroke and many cancerpatients are just a few examples where the use of heparin should beavoided due to the risk of bleeding.

As evidenced herein, administration of a compound of formula Isignificantly inhibited VTE in an in vivo treatment model of thrombusformation under continuous blood flow without an increased bleedingrisk. Effects of the compound in this treatment model are comparable tothe standard of care using LMW heparin. However, LMW heparin is a knownanti-coagulant and delays clotting over four times longer than controlbleeding times. Also as described herein, the compound of formula I onlyslightly delays clotting and is a significant improvement in reducingbleeding time over LMW heparin. Accordingly, the agents described hereinmay be useful when the risk of bleeding is not significant, but also maybe useful in a wide variety of situations when the risk of bleeding issignificant, and particularly when use of anti-thrombosis agents withanti-coagulant properties (such as LMW heparin) is contraindicated.

At least one (i.e., one or more) of the above described agents (i.e., anE-selectin antagonist, such as a glycomimetic compound of formula (I))may be administered in combination with at least one (i.e., one or more)additional anti-thrombosis agent. An agent described herein (i.e., anE-selectin antagonist) may function independent of the anti-thrombosisagent, or may function in coordination with the anti-thrombosis agent.In addition, the administration of one or more of the agents describedherein may be in conjunction with one or more other therapies, e.g., forreducing toxicities of therapy. For example, at least one palliativeagent to counteract (at least in part) a side effect of therapy may beadministered. Agents (chemical or biological) that promote recovery, orcounteract side effects of administration of antibiotics orcorticosteroids, are examples of such palliative agents. At least oneagent described herein may be administered before, after or concurrentlywith administration of at least one additional anti-thrombosis agent orat least one palliative agent to reduce a side effect of therapy. Whereadministration is concurrent, the combination may be administered from asingle container or two (or more) separate containers.

A wide variety of individuals are candidates for treatment as describedherein. Thrombus formation may occur in infants, children, teenagers andadults. An individual may have a hereditary predisposition tothrombosis. Thrombosis may be initiated, for example, due to a medicalcondition (such as cancer or pregnancy), a medical procedure (such assurgery) or an environmental condition (such as prolonged immobility).Other individuals at risk for thrombus formation include those who havepreviously presented with a thrombus.

In particular embodiments of the methods described herein, the subjectis a human or non-human animal. A subject in need of the treatmentsdescribed herein may exhibit symptoms or sequelae of thrombosis disease,disorder, or condition described herein or may be at risk of developingthe disease, disorder, or condition. Non-human animals that may betreated include mammals, for example, non-human primates (e.g., monkey,chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils,hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig, miniaturepig), equine, canine, feline, bovine, and other domestic, farm, and zooanimals.

The effectiveness of a compound, agent, or pharmaceutical compositiondescribed herein in treating or preventing a disease or disorder orcondition described herein, and determining and adjusting an appropriatedosing regimen (e.g., adjusting the amount of compound per dose and/ornumber of doses and frequency of dosing), can readily be determined by aperson of ordinary skill in the medical and clinical arts. One or anycombination of diagnostic methods, including physical examination,assessment and monitoring of clinical symptoms, and performance ofanalytical tests and methods described herein, may be used formonitoring the health status of the subject.

Methods for Characterizing Therapeutic Agents

Characterizing at least one biological activity of a therapeutic agentdescribed herein may be determined by performing one or more in vitroand in vivo studies routinely practiced in the art and described hereinor in the art. In vitro assays include without limitation bindingassays, immunoassays, competitive binding assays and cell based activityassays. Animal model studies may also be performed, which are typicallyrodent animal studies described in the art or routinely developed oradapted by a person skilled in the art to characterize an agent,including determining efficacy, in vivo. Non-human primate animal modelsmay be used in pre-clinical studies that precede clinical studies;however, these animal models are not typically employed in the sameroutine manner as rodent animal studies designed for assessing theeffectiveness or other characteristics of a therapeutic. Persons skilledin the art of design and execution of animal model studies can alsoreadily determine the appropriate control groups to include with thestudies as well as determine the appropriate statistical analysis oranalyses for evaluating the data.

An inhibition assay may be used to screen for antagonists of E-selectin.For example, an assay may be performed to characterize the capability ofa compound or other agent described herein to inhibit (i.e., reduce,block, decrease, or prevent in a statistically of biologicallysignificant manner) interaction of E-selectin with sLe^(a) or sLe^(x).The inhibition assay may be a competitive binding assay, which allowsthe determination of IC₅₀ values. By way of example, the methodcomprises immobilizing E-selectin/Ig chimera onto a matrix (e.g., amulti-well plate, which are typically made from a polymer, such aspolystyrene; a test tube, and the like); adding a composition to reducenonspecific binding (e.g., a composition comprising non-fat dried milkor bovine serum albumin or other blocking buffer routinely used by aperson skilled in the art); contacting the immobilized E-selectin withthe candidate agent in the presence of sLe^(a) comprising a reportergroup under conditions and for a time sufficient to permit sLe^(a) tobind to the immobilized E-selectin; washing the immobilized E-selectin;and detecting the amount of sLe^(a) bound to immobilized E-selectin.Variations of such steps can be readily and routinely accomplished by aperson of ordinary skill in the art.

A person skilled in the art is also familiar with assays and animalmodels to assess whether an E-selectin antagonist is free of significantanti-coagulation properties. For example, an assay that determines thetime required to form a clot may be used to screen for or characterizethe capability of an E-selectin antagonist to significantly delayclotting, wherein an agent that exhibits reduced, absent, or lack ofcapability to delay clotting is desired. By way of example, bleedingtimes may be evaluated in rodents that are injected with a testE-selectin antagonist or a control, and bleeding times recorded after atail vein is nicked and the tail immersed in isotonic saline.

Conditions for a particular assay include temperature, buffers(including salts, cations, media), and other components that maintainthe integrity of any cell used in the assay and the compound, which aperson of ordinary skill in the art will be familiar and/or which can bereadily determined. A person of ordinary skill in the art also readilyappreciates that appropriate controls can be designed and included whenperforming the in vitro methods and in vivo methods described herein.

The source of an agent that is characterized by one or more assays andtechniques described herein and in the art may be a biological samplethat is obtained from a subject who has been treated with the agent. Thecells that may be used in the assay may also be provided in a biologicalsample. A “biological sample” may include a sample from a subject, andmay be a blood sample (from which serum or plasma may be prepared), abiopsy specimen, one or more body fluids (e.g., lung lavage, ascites,mucosal washings, synovial fluid, urine), bone marrow, lymph nodes,tissue explant, organ culture, or any other tissue or cell preparationfrom the subject or a biological source. A biological sample may furtherrefer to a tissue or cell preparation in which the morphologicalintegrity or physical state has been disrupted, for example, bydissection, dissociation, solubilization, fractionation, homogenization,biochemical or chemical extraction, pulverization, lyophilization,sonication, or any other means for processing a sample derived from asubject or biological source. In certain embodiments, the subject orbiological source may be a human or non-human animal, a primary cellculture (e.g., immune cells), or culture adapted cell line, includingbut not limited to, genetically engineered cell lines that may containchromosomally integrated or episomal recombinant nucleic acid sequences,immortalized or immortalizable cell lines, somatic cell hybrid celllines, differentiated or differentiatable cell lines, transformed celllines, and the like.

Exemplary animal models are described herein and in the art fordetermining the effectiveness of an E-selectin antagonist. Numerouscancer animal models are routinely practiced in the art. By way ofnon-limiting examples, models of ALL, multiple myeloma, AML and solidtumor cancer models are available for determining the effectiveness ofan E-selectin antagonist. Typically, animals are engrafted with a tumorcell line (such as without limitation, a pancreatic, breast, colon,ovarian, ALL, AML, multiple myeloma tumor cell line) and an agent ofinterest is administered prior to engraftment, during tumor growth,and/or after a tumor has been established. Numerous statistical analysesare available and understood by a person skilled in the art and may beapplied to compare the effect of an agent to one or more appropriatecontrols.

Pharmaceutical Compositions and Methods of Using PharmaceuticalCompositions

Also provided herein are pharmaceutical compositions that comprise anyone or more of the E-selectin antagonist agents described herein, suchas one or more of the glycomimetic compounds of formula I (andsubstructures and specific structures thereof) described herein. Thecompounds, isolated antibodies and other E-selectin antagonistsdescribed herein may also be prepared for pharmaceutical use in asubject, including a human subject. The compounds described herein maybe formulated in a pharmaceutical composition for use in treatment orpreventive (or prophylactic) treatment (e.g., reducing the likelihood ofoccurrence or of exacerbation of a disease, or of one or more symptomsof the disease). The methods and excipients described herein areexemplary and are in no way limiting.

In pharmaceutical dosage forms, any one or more of the glycomimeticcompounds of formula I, substructures and specific structures describedherein may be administered in the form of a pharmaceutically acceptablederivative, such as a salt, or they may also be used alone or inappropriate association, as well as in combination, with otherpharmaceutically active compounds. By way of example, as describedherein with respect to methods of use, an E-selectin antagonist may beadministered to a subject who is also receiving chemotherapy,radiotherapy, a combination or chemotherapy and radiotherapy.

An effective amount or therapeutically effective amount refers to anamount of a glycomimetic compound or a composition comprising one ormore compounds; or one or more isolated antibodies (or other E-selectinantagonist agent), that when administered to a subject, either as asingle dose or as part of a series of doses, is effective to produce adesired therapeutic effect. Optimal doses may generally be determinedusing experimental models and/or clinical trials. Design and executionof pre-clinical and clinical studies for each of the therapeutics(including when administered for prophylactic benefit) described hereinare well within the skill of a person of ordinary skill in the relevantart. The optimal dose of a therapeutic may depend upon the body mass,weight, or blood volume of the subject. In general, the amount of acompound described herein, that is present in a dose, ranges from about0.01 μg to about 1000 μg per kg weight of the host. In general, theamount of a polypeptide or peptide, or an antibody or antigen-bindingfragment thereof, as described herein, present in a dose, also rangesfrom about 0.01 μg to about 1000 μg per kg of subject. The use of theminimum dose that is sufficient to provide effective therapy is usuallypreferred. Subjects may generally be monitored for therapeuticeffectiveness using assays suitable for the disease or condition beingtreated or prevented, which assays will be familiar to those havingordinary skill in the art and are described herein. The level of acompound or polypeptide that is administered to a subject may bemonitored by determining the level of the compound, peptide, antibody orantigen-binding fragment thereof, or polypeptide (or a metabolite of anyof the aforementioned molecules) in a biological fluid, for example, inthe blood, blood fraction (e.g., serum), and/or in the urine, and/orother biological sample from the subject. Any method practiced in theart to detect the molecule may be used to measure the level of themolecule during the course of a therapeutic regimen.

The dose of a compound, peptide, antibody or antigen-binding fragmentthereof, or polypeptide described herein may depend upon the subject'scondition, that is, stage of the disease, severity of symptoms caused bythe disease, general health status, as well as age, gender, and weight,and other factors apparent to a person of ordinary skill in the medicalart. Similarly, the dose of the therapeutic for treating a disease ordisorder may be determined according to parameters understood by aperson of ordinary skill in the medical art.

Pharmaceutical compositions may be administered in a manner appropriateto the disease or disorder to be treated as determined by persons ofordinary skill in the medical arts. An appropriate dose and a suitableduration and frequency of administration will be determined by suchfactors as discussed herein, including the condition of the patient, thetype and severity of the patient's disease, the particular form of theactive ingredient, and the method of administration. In general, anappropriate dose (or effective dose) and treatment regimen provides thepharmaceutical composition(s) as described herein in an amountsufficient to provide therapeutic and/or prophylactic benefit (forexample, an improved clinical outcome, such as more frequent complete orpartial remissions, or longer disease-free and/or overall survival, or alessening of symptom severity or other benefit as described in detailabove).

The pharmaceutical compositions described herein may be administered toa subject in need thereof by any one of several routes that effectivelydeliver an effective amount of the compound. Such administrative routesinclude, for example, topical, oral, nasal, intrathecal, enteral,buccal, sublingual, transdermal, rectal, vaginal, intraocular,subconjunctival, sublingual or parenteral administration, includingsubcutaneous, intravenous, intramuscular, intrasternal, intracavernous,intrameatal or intraurethral injection or infusion. Compositionsadministered by these routes of administration and others are describedin greater detail herein.

A pharmaceutical composition may be a sterile aqueous or sterilenon-aqueous solution, suspension or emulsion, which additionallycomprises a physiologically acceptable excipient (pharmaceuticallyacceptable or suitable excipient or carrier) (i.e., a non-toxic materialthat does not interfere with the activity of the active ingredient).Such compositions may be in the form of a solid, liquid, or gas(aerosol). Alternatively, compositions described herein may beformulated as a lyophilizate, or compounds and polypeptides or peptidesdescribed herein may be encapsulated within liposomes using technologyknown in the art. Pharmaceutical compositions may also contain othercomponents, which may be biologically active or inactive. Suchcomponents include, but are not limited to, buffers (e.g., neutralbuffered saline or phosphate buffered saline), carbohydrates (e.g.,glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptidesor amino acids such as glycine, antioxidants, chelating agents such asEDTA or glutathione, stabilizers, dyes, flavoring agents, and suspendingagents and/or preservatives.

Any suitable excipient or carrier known to those of ordinary skill inthe art for use in pharmaceutical compositions may be employed in thecompositions described herein. Excipients for therapeutic use are wellknown, and are described, for example, in Remington: The Science andPractice of Pharmacy (Gennaro, 21^(st) Ed. Mack Pub. Co., Easton, Pa.(2005)). In general, the type of excipient is selected based on the modeof administration, as well as the chemical composition of the activeingredient(s). Pharmaceutical compositions may be formulated for anyappropriate manner of administration, including, for example, topical,oral, nasal, intrathecal, enteral, buccal, sublingual, transdermal,rectal, vaginal, intraocular, subconjunctival, sublingual or parenteraladministration, including subcutaneous, intravenous, intramuscular,intrasternal, intracavernous, intrameatal or intraurethral injection orinfusion. For parenteral administration, the carrier preferablycomprises water, saline, alcohol, a fat, a wax or a buffer. For oraladministration, any of the above excipients or a solid excipient orcarrier, such as mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, kaolin, glycerin, starch dextrins, sodiumalginate, carboxymethylcellulose, ethyl cellulose, glucose, sucroseand/or magnesium carbonate, may be employed.

A pharmaceutical composition (e.g., for oral administration or deliveryby injection) may be in the form of a liquid. A liquid pharmaceuticalcomposition may include, for example, one or more of the following: asterile diluent such as water for injection, saline solution, preferablyphysiological saline, Ringer's solution, isotonic sodium chloride, fixedoils that may serve as the solvent or suspending medium, polyethyleneglycols, glycerin, propylene glycol or other solvents; antibacterialagents; antioxidants; chelating agents; buffers and agents for theadjustment of tonicity such as sodium chloride or dextrose. A parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic. The use of physiological saline ispreferred, and an injectable pharmaceutical composition is preferablysterile.

For oral formulations, at least one of the E-selectin antagonist agentsdescribed herein can be used alone or in combination with appropriateadditives to make tablets, powders, granules or capsules, for example,with any one or more conventional additives, disintegrators, lubricants,and if desired, diluents, buffering agents, moistening agents,preservatives, coloring agents, and flavoring agents. The compositionsmay be formulated to include a buffering agent to provide for protectionof the active ingredient from low pH of the gastric environment and/oran enteric coating. A composition may be formulated for oral deliverywith a flavoring agent, e.g., in a liquid, solid or semi-solidformulation and/or with an enteric coating.

Oral formulations may be provided as gelatin capsules, which may containthe active compound or biological along with powdered carriers. Similarcarriers and diluents may be used to make compressed tablets. Tabletsand capsules can be manufactured as sustained release products toprovide for continuous release of active ingredients over a period oftime. Compressed tablets can be sugar coated or film coated to mask anyunpleasant taste and protect the tablet from the atmosphere, or entericcoated for selective disintegration in the gastrointestinal tract.

A pharmaceutical composition may be formulated for sustained or slowrelease. Such compositions may generally be prepared using well knowntechnology and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain the active therapeuticdispersed in a carrier matrix and/or contained within a reservoirsurrounded by a rate controlling membrane. Excipients for use withinsuch formulations are biocompatible, and may also be biodegradable;preferably the formulation provides a relatively constant level ofactive component release. The amount of active therapeutic containedwithin a sustained release formulation depends upon the site ofimplantation, the rate and expected duration of release, and the natureof the condition to be treated or prevented.

The pharmaceutical compositions described herein can be formulated assuppositories by mixing with a variety of bases such as emulsifyingbases or water-soluble bases. The pharmaceutical compositions may beprepared as aerosol formulations to be administered via inhalation. Thecompositions may be formulated into pressurized acceptable propellantssuch as dichlorodifluoromethane, propane, nitrogen and the like.

Any one or more of the E-selectin antagonist agents described herein maybe administered topically (e.g., by transdermal administration). Topicalformulations may be in the form of a transdermal patch, ointment, paste,lotion, cream, gel, and the like. Topical formulations may include oneor more of a penetrating agent or enhancer (also call permeationenhancer), thickener, diluent, emulsifier, dispersing aid, or binder.Physical penetration enhancers include, for example, electrophoretictechniques such as iontophoresis, use of ultrasound (or“phonophoresis”), and the like. Chemical penetration enhancers areagents administered either prior to, with, or immediately followingadministration of the therapeutic, which increase the permeability ofthe skin, particularly the stratum corneum, to provide for enhancedpenetration of the drug through the skin. Additional chemical andphysical penetration enhancers are described in, for example,Transdermal Delivery of Drugs, A. F. Kydonieus (ED) 1987 CRL Press;Percutaneous Penetration Enhancers, eds. Smith et al. (CRC Press, 1995);Lenneräs et al., J. Pharm. Pharmacol. 54:499-508 (2002); Karande et al.,Pharm. Res. 19:655-60 (2002); Vaddi et al., Int. J. Pharm. 91:1639-51,(2002); Ventura et al., J. Drug Target 9:379-93 (2001); Shokri et al.,Int. J. Pharm. 228 (1-2):99-107 (2001); Suzuki et al., Biol. Pharm.Bull. 24:698-700 (2001); Alberti et al., J. Control Release 71:319-27(2001); Goldstein et al., Urology 57:301-5 (2001); Kiijavainen et al.,Eur. J. Pharm. Sci. 10:97-102 (2000); and Tenjarla et al., Int. J.Pharm. 192:147-58 (1999).

Kits with unit doses of one or more of the compounds, polypeptides,peptides, aptamers, antibodies and antigen binding fragments, thereofdescribed herein, usually in oral or injectable doses, are provided.Such kits may include a container containing the unit dose, aninformational package insert describing the use and attendant benefitsof the therapeutic in treating the pathological condition of interest,and optionally an appliance or device for delivery of the composition.

EXAMPLES Example 1 Synthesis of E-Selectin Inhibitor

Exemplary glycomimetic compounds of formula I were synthesized asdescribed in this Example and as shown in the exemplary synthesisschemes set forth in FIGS. 1-2.

Synthesis of Compound 2

Compound 1 (60 g) was suspended in H₂O (800 ml) and cooled to 0° C.Solid NaHCO₃ (120 g) was added in portion with stirring and then asolution of KI (474.3 g) and I₂ (127 g) in H₂O (800 ml) was added withstirring. Reaction mixture was stirred at room temperature overnight inthe dark. Reaction mixture was then extracted with CH₂Cl₂ (3×500 ml).The organic layer was washed with Na₂S₂O₃ solution (2×500 ml) and thenthe combined aqueous layers were extracted with CH₂Cl₂ (2×300 ml).Organic layers (2100 ml)were combined and washed with cold H₂O (1×500ml) and cold brine (1×500 ml). The organic layer was dried over Na₂SO₄,filtered, and concentrated to dryness to give compound 2 as light yellowcrystals (119 g). Purity: >95% by TLC.

Synthesis of Compound 3

To a solution of compound 2 (119 g) in THF (1600 ml) was added DBU (119ml) with stirring at room temperature and the reaction mixture wasgently refluxed overnight with stirring. Some precipitate forms and TLCshowed no starting material left. The reaction mixture was concentratedto dryness and dissolved in EtOAc (300 ml), washed with 0.5 M HCl (200ml) until pH 2-3 of the aqueous wash, and then the organic layer wasfurther washed with H₂O (200 ml). Aqueous layers were combined andextracted with EtOAc (3×200 ml) to produce a second organic layer.Combined organic layers (900 ml) were washed with brine, dried (Na₂SO₄),filtered and concentrated to dryness to give compound 3 (58 g).Purity: >95% by TLC.

Synthesis of Compound 4

To a solution of compound 3 (58 g) in MeOH (800 ml) was added NaHCO₃ (47g) with stirring. The reaction mixture was stirred under gentle refluxfor 3 h, cooled to room temperature, filtered and concentrated todryness. The residue was dissolved in EtOAc (300 ml) and washed withH₂O. Aqueous layer was extracted with EtOAc (3×100 ml). Combined organiclayers (600 ml) were washed with 0.5M HCl (200 ml), H₂O (100 ml), andbrine (100 ml), dried (Na₂SO₄), filtered, and concentrated to dryness.The residue was purified by column chromatography (SiO₂, Hexanes-EtOAc3:1→3:2) to give compound 4 (54 g). Purity: >95% by TLC.

Synthesis of Compound 5

Compound 4 (31 g) was dissolved in tBuOMe (620 ml) and vinylacetate (166ml) added with vigorous stirring. Novozyme 435 (1.4 g) was added andvigorous stirring continued for 5.5 h. The reaction mixture was filteredand stored at −20° C. After 12-18 hours, another batch of Novozyme 435resin (1.4 g) was added and stirred vigorously for 8 h. Resin wasfiltered and concentrated to dryness. Oily residue was purified byCombiFlash® system (silica) using 0→50% EtOAc/Hexanes to give compound 5(13.0 g).

Synthesis of Compound 6

Compound 5 (13.5 g) was dissolved in CH₂Cl₂ (300 ml) under argon andTBDMS-CI (26.4 g) added with stirring at room temperature under argon.DBU (32.4 ml) was added and stirring continued for overnight at roomtemperature under argon. MeOH (30 ml) was added and washed with coldsaturated solution of NaHCO₃ (200 ml), brine (150 ml). The organic layerwas dried (Na₂SO₄), filtered and concentrated to dryness. The residuewas purified by CombiFlash® system (SiO₂) using solvent EtOAc-Hexanes(0-15%) to give compound 6 (18 g). Purity >95% by TLC.

Synthesis of Compound 7

Compound 6 (12 g) was dissolved in CH₂Cl₂ (400 ml) and cooled to 0° C.m-chloroperbenzoic acid (77%, 19 g) was added and the solution stirredfor few hours during which the temperature of the reaction mixturereached to room temperature. The stirring was continued overnight atroom temperature. CH₂Cl₂ (300 ml) was added and washed with coldsaturated solution of NaHCO₃ (3×400 ml), brine (cold), dried (Na₂SO₄),filtered, and concentrated to dryness. The residue was purified byCombiFlash® system (SiO₂) using EtOAc-Hexanes (0→30%) to give 7 (9 g).Purity: >95% by TLC.

Synthesis of Compound 8

All operation of this step was done in argon atmosphere. CuCN (9.42 g)was dried at 160° C. under vacuum for 40 min, cooled down to roomtemperature and suspended in THF (80 ml). The mixture was cooled down to−78° C. During this time, tetravinyltin (12 ml) and n-BuLi in hexane(2.5 M, 100 ml) were reacted for 30 min at 0° C. in THF (30 ml). Thissolution was added to the mixture of CuCN in THF, and the resultingmixture was stirred for 30 min. at −20° C. The mixture was then cooledto −78° C. and to which was added a solution of freshly distilledBF₃.Et2O (6 ml) in THF (20 ml). The mixture was stirred for 20 min. at−78° C. Compound 7 (5 g) in THF (40 ml) was added and the reactionmixture was stirred at −78° C. for 5 h. MeOH (7 ml) and Et₃N (3 ml) wasadded and the mixture was concentrated to dryness. The residue wasdissolved in EtOAc (200 ml) and washed with saturated solution of NaHCO₃(2×100 ml), brine (100 ml), dried (Na₂SO₄), filtered, and concentratedto dryness. The residue was purified by CombiFlash® system (SiO₂) usingsolvent EtOAc-Hexanes (0→5%) to give compound 8 (2.5 g).

Synthesis of Compound 10

Compound 8 (2.25 g, 7 mmol) was dissolved in toluene (7 ml), and solventevaporated off. The process was repeated twice and finally dried undervacuum for 15 min. The residue was dissolved in anhydrous CH₂Cl₂ (45 ml)and DMF (45 ml) was added. The solution was stirred under argon at roomtemperature and molecular sieves (3 g, 4 Å, powdered and flamed dried)added. Et₄NBr (3.3 g, 15.7 mmol, 2.2 equivalents, dried at 200° C. for 2h) was added and the stirring continued for 1 h at room temperatureunder argon.

Compound 9 (5.13 g, 10 mmol, 1.42 equivalents) was co-evaporated withtoluene (3×20 ml), dried under vacuum, and dissolved in CH₂Cl₂ (45 ml).The reaction mixture was placed in an ice-bath and stirred for 10 min.To this solution was added Br₂ (0.8 ml, 15 mmol, 1.5 equivalents)drop-wise with stirring in the ice-bath. Stirring was continued for 40min at the same temperature. The ice-bath was removed and cyclohexene(2.1 ml) added slowly with stirring after 10 min. The reaction mixturewas stirred for 10 min. and added slowly to the reaction mixture abovewith stirring at room temperature under argon. Stirring continued for 17h and then pyridine (4 ml) was added, filtered and the filtrateconcentrated to dryness. The residue was dissolved in CH₂Cl₂ (100 ml)and transferred to a separatory funnel. The organic layer was washedwith cold brine (2×75 ml), dried (Na₂SO₄), filtered and concentrated todryness, co-evaporated with toluene (3×50 ml), and dried under vacuum.The residue was dissolved in THF (8 ml) and a solution of TBAF (1 M inTHF, 10 ml, 10 mmol, 1.42 equivalents) added with stirring at roomtemperature. Stirring was continued for 15 h and solvent evaporated off.The residue was dissolved in CH₂Cl₂ (100 ml) and transferred to aseparatory funnel, washed with cold brine (2×75 ml), dried (Na₂SO₄),filtered, and concentrated to dryness. The residue was purified bycolumn chromatography (Hexanes-Ethyl acetate from 100% hexanes to 70%hexanes in EtOAc) to give compound 10 (1.6 g, 2.59 mmol, 37% overall intwo steps). TLC: 5% EtOAc in hexanes and 33% EtOAc in hexanes.

Synthesis of Compound 12

Commercially available compound 11 (10 g) was dried overnight undervacuum overnight and added to a solution of NaOMe (5M, 10 ml) in MeOH(200 ml) with stirring at room temperature under argon. Stirring wascontinued for overnight at room temperature argon, and Et₃N (7 ml) wasadded followed by allylchloroformate (3.5 ml) dropwise. Stirring wascontinued for 6 h at room temperature under argon. The reaction mixturewas concentrated to dryness and dissolved in pyridine (100 ml). AC₂O (50ml) was added at room temperature under argon and stirred at roomtemperature for overnight. The reaction mixture was concentrated todryness and purified by column chromatography on CombiFlash® systemusing EtOAc-Hexanes (0-100%). The desired fractions were collected andconcentrated to dryness to give Compound 12(10.2 g).

Synthesis of Compound 13

Compound 12 (7.5 g) was dissolved in DMF (140 ml) to which was addedNH₄OAC (4.05 g) with stirring. Stirring was continued for overnight atroom temperature under argon. The next day the reaction mixture wasstirred at 50° C. under argon for 8 h. The reaction mixture wasconcentrated to dryness and the residue dissolved in EtOAc (150 ml),washed with brine (100 ml), dried (Na₂SO₄), filtered, and concentratedto dryness. The residue was purified by column chromatography (SiO₂,Hexanes-EtOAc 2:1→1:2) to give Compound 13 (6 g).

Synthesis of Compound 14

Compound 13 (6 g) was dissolved in CH₂Cl₂ (50 ml) to which was addedCCl₃CN (6 ml) and DBU (0.5 ml). The reaction mixture was stirred at roomtemperature for 0.5 h, solvent was evaporated off and the residue waspurified by column chromatography (silica gel) to give Compound 14 (4.5g).

Synthesis of Compound 15

Compound 10 (2g) and compound 14(2.1 g) was dissolved in CH₂Cl₂ (40 ml).To this solution were added molecular sieves (4 Å, 0.8 g) and stirred atroom temperature for 30 min. The solution was then cooled to 0° C. andBF₃Et₂O (0.25 ml dissolved in 5 ml) is added with stirring at 0° C. Thereaction mixture was stirred at 0°C. for 2 h. Et₃N (0.5 ml) was addedand the solvent was evaporated off. The residue was purified by columnchromatography (silica gel) to give Compound 15 (1.8 g).

Synthesis of Compound 16

Compound 15 (1.7 g) was treated with 0.01 N NaOMe in MeOH (10 ml) for 2h and neutralized with IR-120 (H⁺) resin, filtered, and concentrated todryness to give Compound 16 (1.25 g).

Synthesis of Compound 17

To a solution of compound 16 (1.2 g) in CH₃CN (30 ml) was added Et3N(0.28 ml) and cooled to 0° C. To this solution was added BzCN (0.35 mgin 10 ml CH₃CN) dropwise during 20 min at 0° C. The reaction mixture wasstirred for 1 h at 0° C. and concentrated to dryness. The residue waspurified by column chromatography (silica gel) to give compound 17 (0.95g).

Synthesis of Compound 19

Compound 17 (0.9 g) was dissolved in MeOH (12 ml). To this solution wasadded Bu₂SnO (0.4 g) and the mixture was refluxed for2 h. Solvent wasevaporated off and the residual solvent was co-evaporated off withtoluene 3 times. The residue was dissolved in dimethoxy ethane (15 ml).To this solution was added CsF (0.8 g) and compound 18 (2.1 g,synthesized as described previously, J. Med. Chem. 42:4909, 1999). Thereaction mixture was stirred overnight at room temperature, and thesolvent was evaporated off. The residue was purified by columnchromatography to give compound 19 (0.8 g).

Synthesis of Compound 20

Compound 19 (0.7g) was dissolved in CH₂Cl₂ (20 ml). To this solution wasadded Pd(Ph)4 (0.14 g), Bu₃SnH (0.15 ml), and Ac2O (0.3 ml) and thereaction mixture is stirred at room temperature for 1 h. Solvent wasevaporated off and the residue was purified by column chromatography(silica gel) to give compound 20 (0.5 g).

Synthesis of Compound 21

To a solution of compound 20 (0.45 g) in dioxane-H₂O-AcOH (10:2:1, 2.6ml) was added 10% Pd—C (0.15 g), and the reaction mixture was shaken atroom temperature under positive pressure (20 psi) of hydrogen for 5 h.The solid was filtered off, and the filtrate was concentrated todryness. The residue was purified by column chromatography (silica gel)to give Compound 21 (0:3 g).

Synthesis of Compound 22

Compound 21 (0.28 g) was treated with 0.025 N NaOMe in MeOH (5 ml) for 4h, neutralized with IR-120 (H+) resin, filtered, and the filtrate wasconcentrated to dryness to give compound 22 (0.21 g).

Synthesis of Compound 23

Compound 22 (0.18 g) was dissolved in ethylenediamine (2 ml) and stirredat 80° C. for 8 h. Solvent was evaporated off and the residue purifiedusing Sep-pak C18 cartridges to give compound 23 (0.15 g).

Synthesis of Compound 25

Compound 23 (200 mg) was dissolved into 1 mL DMF. To this solution wasadded commercially available compound 24 (400 mg). Triethyl amine (100μL) was added dropwise to the react reaction mixture to adjust the pH to10. The reaction mixture was stirred at room temperature for 1 h. Afterevaporation to dryness, the residue was purified by HPLC to affordcompound 25 (200 mg). See FIG. 1D.

Synthesis of Compound 45

Compound 25 (300 mg) was dissolved into 3 mL DMF. Diisopropylethylamine(60 μL) and HATU (131 mg) were added at room temperature. After stirringfor 5 minutes, dimethylamine (2.3 mL, 2M solution in THF) was addeddropwise. The reaction was stirred at room temperature for 1 hour. Thereaction mixture was concentrated to dryness in vacuo. The residue wasdissolved in water and loaded onto a 10 g C-18 cartridge. Elution withwater followed by 1/1 water/MeOH afforded compound 45 (100 mg). m/zcalculated for C₆H₁₁₄N₄O₂₆=1330.8. Found=1353.6 (M+Na). ¹H NMR 400 MHz(D₂O, set at 4.80 ppm) δ 0.87 (t, J=7.6 Hz, 3H), 0.94-0.99 (m, 2H),1.20-1.25 (m, 4H), 1.25 (d, J=6.4 Hz, 3H), 1.26-1.45 (m, 4H), 1.52-1.73(m, 6H), 1.79-1.88 (m, 3H), 2.00 (s, 3H), 2.11-2.19 (br d, 1H), 2.33(tt,J=12.4 Hz, J=3.2 Hz, 1H), 2.53 (t, J=6.4 Hz, 2H), 2.95 (s, 3H) 3.06 (s,3H), 3.28 (t, J=12.5 Hz, 1H), 3.31-3.38 (m, 8H), 3.51-3.54 (m, 2H), 3.61(dd, J=8.0 Hz, J=0.8 Hz, 1H), 3.63 (dd, J=8.0 Hz, J=2.0 Hz, 1H), 3.70(s, 44H), 3.73-3.76 (m, 1H), 3.78 (t, J=6.0 Hz, 1H), 3.81-3.82 (m, 1H),3.88 (dd, J=3.6 Hz, 1H), 3.99 (bs, 1H), 4.54 (dd, J=8.8 Hz, J=2.0 Hz,2H) 4.91 (q, J=6.8 Hz, 1H), 5.04 (d, J=3.6 Hz, 1H).

Synthesis of Compound 26

Compound 26 was synthesized as described for compound 25 (see FIG. 1D)except that the PEG reactant had an n of 8 (i.e., 8 repeating PEG units)rather than 12 as for the synthesis of compound 25.

m/z calculated for C₅₂H₉₃N₃O₂₃=1127.6. Found=1151.6 (M+Na). ¹H NMR 600MHz (D₂O, set at 4.67 ppm) d 0.71 (t, J=7.2 Hz, 3H), 0.76 (br quin,J=12.0 Hz, 2H), 0.99-1.06 (m, 4H), 1.08 (d, J=6.6 Hz, 3H), 1.15-1.19 (brquin, J=6.6 Hz, 1H), 1.21-1.25 (m, 2H), 1.39-1.48 (m, 5H), 1.50-1.60 (m,3H), 1.70 (br d, J=10.2 Hz, 2H), 1.91 (s, 3H), 1.99 (m, 1H), 2.16 (br t,J=12.6 Hz, 1H), 2.36 (t, J=6 Hz, 2H), 3.11-3.15 (m, 2H), 3.18 (t, J=9.6Hz, 3H), 3.22 (s, 3H), 3.38 (dd, J=7.8 Hz, J=4.2 Hz, 2H), 3.46 (dd,J=4.2 Hz, 1H), 3.47 (s, 1H), 3.52-3.55 (m 27H), 3.56-3.59 (m, 3H),3.61-3.64 (m, 3H), 3.65 (d, J=3.6 Hz, 1H), 3.72 (dd, J=10.2 Hz, J=3.0Hz, 1H), 3.80 (d, J=2.4 Hz, 1H), 3.85 (br s, 1H), 3.94 (dd, J=9.6 Hz,J=3.6 Hz, 1H), 4.36 (br s, 1H), 4.77 (q, J=6.6 Hz, 1H), 4.88 (d, J=4.2Hz, 1H).

Synthesis of Compound 27

Compound 27 was synthesized as described in FIG. 2.

Synthesis of Compound 27A

Compound 19 (0.05 g) was dissolved in CH₂Cl₂ (10 ml). To this solutionwas added Pd[(Ph₃)P]₄ (5 mg), Bu3SnH (0.0011 ml), and (CF3CO)₂O (0.0015ml) with stirring at room temperature. Stirring was continued for 30 minat room temperature. The reaction mixture was evaporated to drynessunder reduced pressure and the residue was purified by columnchromatography (silica gel) to give compound 27A (0.030 g).

Compound 27A (0.025 g) was subjected to hydrogenation with 10% Pd—Cexactly in same way as described for compound 21 and the solvent wasevaporated off after filtering of the catalyst. The residue was treatedwith NaOMe in MeOH as described for compound 22, neutralized with IR-120(H+) resin, filtered, and the solvent was evaporated off. The residuewas purified by reverse phase (C18) HPLC to give compound 27 (7 mg). m/zcalculated for C₃₃H₅₂F₃NO₁₅=759.3. Found=782.3 (M+Na).

Synthesis of Compound 28

Synthesis Scheme for Compound 28

Synthesis of Compound 28

Commercially available compound 27B (0.014 g) was dissolved in DMF (1ml). To this solution was added DIPEA (0.00175 ml) and HATU (0.038 g)and the reaction mixture was stirred for 2 min at room temperature.Compound 23 (0.035 g) was added and the reaction mixture was stirred for1 h at room temperature. Solvent was evaporated off and the residue waspurified by HPLC (C18) to give compound 28 (17 mg).

Synthesis of Compound 29

Synthesis Scheme for Compound 29

Commercially available compound 27C (0.021 g) was reacted with compound23 (0.035 g) exactly in the same way as described for compound 28 andpurified by HPLC (C18) to give compound 29 (0.020 g).

Example 2 E-Selectin Activity-Binding Assay

The inhibition assay to screen for and characterize glycomimeticantagonists of E-selectin is a competitive binding assay, which allowsthe determination of IC₅₀ values. E-selectin/Ig chimera was immobilizedin 96 well microtiter plates by incubation at 37° C. for 2 hours. Toreduce nonspecific binding, bovine serum albumin was added to each welland incubated at room temperature for 2 hours. The plate was washed andserial dilutions of the test compounds were added to the wells in thepresence of conjugates of biotinylated, sLe^(a) polyacrylamide withstreptavidin/horseradish peroxidase and incubated for 2 hours at roomtemperature.

To determine the amount of sLe^(a) bound to immobilized E-selectin afterwashing, the peroxidase substrate, 3,3′,5,5′ tetramethylbenzidine (TMB)was added. After 3 minutes, the enzyme reaction was stopped by theaddition of H₃PO₄, and the absorbance of light at a wavelength of 450 nmwas determined. The concentration of test compound required to inhibitbinding by 50% was determined and reported as the IC₅₀ value for eachglycomimetic E-selectin antagonist as shown in the table below. IC₅₀values for exemplary compounds disclosed herein are provided in thefollowing table.

E-Selectin Antagonist Activity of Glycomimetic Compounds Compound IC₅₀(μM) 22 <4.0 27 <4.0 29 <4.0 25 <4.0 28 <4.0 45 <4.0

In addition to reporting the absolute IC₅₀ value as measured above,relative IC₅₀ values (rIC₅₀) are determined by a ratio of the IC₅₀measured for the test compound to that of an internal control(reference) stated for each assay.

Substitution of the methyl group at the R³ position of compound 22 witha trimethylfluoro (—CF₃) group did not significantly alter theE-selectin antagonist activity of compound 22; however, the substitutiondid increase the hydrophobicity of the molecule, thereby improving thebioavailability of the glycomimetic compound.

Example 3 Effects of Treatment With an E-Selectin-Specific Antagonist(Compound 25) in a Leukemia Animal Model

The E-selectin ligand HCELL (hematopoietic cell E-/L-selectin ligand) isexpressed by normal hematopoietic stem cells (Merzaban et al., Blood118(7):1774-83 (2011)) as a functional glycoform of CD44. High levelCD44 expression (99%±1.4%) has also been observed by blasts from 55patients with acute myeloid leukemia (AML) (i.e., AML blasts) and byputative CD34³⁰CD38⁻CD123⁺ leukemia stem cells (LSCs) (99.8%±0.6%). Themean fluorescence intensity (MFI) for CD44 expression by AML blasts wasone to two logs higher than the MFI for 16 other adhesion receptors. Themajority of blasts from patients with AML also express an E-selectinligand by flow cytometry: >75%% of 22 primary gated blast samplesexhibit ≧10% binding of E selectin-IgG chimeric protein with a mean of22.7%±0.17% SD, range 1.8 to 66.2%. The ligand was identified as HCELLby immunoprecipitation of CD44 from AML cell membranes, followed bystaining with HECA 452 antibody that recognizes a functionaltrisaccharide domain shared by sialyl Le^(a) and sialyl Le^(x) and isknown to bind to E-selectin. HECA 452 detected the functional glycoformof CD44 known as HCELL, a major ligand for E-selectin and alsoidentified the human lymphocyte homing receptor CLA (cutaneouslymphocyte antigen).

HECA 452 labeled 5 of 6 patient leukemia blast populations, with meanexpression 59.0%±24.8%. HECA 452 antibody labeled CD34⁺CD38⁻CD123⁺ LSCsin addition to leukemic blasts with a higher percent expression in mostcases for the LSCs than the corresponding unfractionated blastpopulation. HECA 452 also labeled 94% of human AML cells that had beenserially engrafted in NODscid IL2Rgc^(−/−) animals, fulfilling thefunctional definition of LSCs (scid repopulating cells), suggesting thatHCELL may be enriched on LSCs. A change in morphology of AML blasts wasobserved when the cells bound to E-selectin coated plastic. The AMLblasts elongated and became more cuboidal and less reflective incontrast to the non-adherent cells, which remained round and refractile.AML blasts appear to bind to the elongated ends of spindle shapedendothelial cells. Compound 25 (concentration 20 μM) inhibited adhesionof primary human AML cells to E-selectin by an average of 45.0%±9.1%SDfor samples from all patients. For one patient for example, the percentinhibition with Compound 25 compared to media control was 33.4%±15.3%SD, p=0.000018.

Adhesion to E-selectin did not confer adhesion-mediated chemotherapyresistance to daunorubicin or cytarabine observed with adhesion torecombinant fibronectin peptide or immobilized VCAM-1 (see Becker etal., Blood 113(4):866-74 (2009)). We demonstrated that a glycomimeticcompound dual inhibitor of E-selectin and CXCR4 (see U.S. PatentApplication Publication No. 2010/0279965) mobilized human AML engraftedin NODscid IL2Rgc^(−/−) mice (see Chien et al., Abstract 579, atAmerican Society for Hematology, 53^(rd) ASH Annual Meeting andExposition, San Diego, Calif.; Dec. 10-13, 2011; Blood, Volume 118,Issue 21) to a greater degree than we observed with CXCR4inhibitorplerixafor (see Chien et al Abstract 1432, at American Society forHematology, 53^(rd) ASH Annual Meeting and Exposition, San Diego,Calif.; Dec. 10-13, 2011) alone (3-4 fold vs. approximately 2 fold). TheE-selectin antagonist Compound 25 (40 mg/kg) mobilized both human andmurine cells in immunodeficient xenograft mice engrafted with human AML.A 2-fold increase in WBC (p=0.00067) and 2-fold increase in human AMLcells (p=0.14) was observed at 3 hrs.

In another experiment, mice were engrafted with human AML blasts andtreated with Compound 25 in combination with daunorubicin and cytarabine(araC) or with daunorubicin and cytarabine only. When the AML cellscomprised about 10% of cells in the blood, the mice were treated. AtDays 1, 2, and 3, groups of animals (four per group) Compound 25 wasadministered at 40 mg/kg twice daily. On Day 1, three hours after thefirst treatment of the animals with Compound 25, daunorubicin (3 mg/kg)and araC (300 mg/kg) were administered. On Days 2 and 3, three hoursafter the first dose of Compound 25, only araC (300 mg/kg) wasadministered. Compound 25 and the chemotherapeutic drugs wereadministered intraperitoneally. A second group of mice receiveddaunorubicin and araC only. Tumor burden was measured in the bonemarrow, blood, and spleen.

The combination of Compound 25 with the two chemotherapeutics resultedin greater depletion of human AML from the bone marrow (22% as many AMLcells) and spleen (31% as many AML cells) than observed withdaunorubicin and cytarabine in the absence of Compound 25. Without beingbound by any particular theory, residence of human AML in the bonemarrow vascular niche may involve E-selectin, and migration of AMLblasts may involve interactions with the vascular endothelium throughE-selectin. See also Chien et al., Poster 4092 at American Society forHematology, 54^(th) ASH Annual Meeting and Exposition, Atlanta, Ga. Dec.8-11, 2012; Blood, Volume 120, Issue 21.

Example 4 Effects of Treatment With an E-Selectin-Specific Antagonist ina Acute Lymphoblastic Leukemia (ALL) Animal Model

The effectiveness of an E-selectin antagonist in an ALL animal model isdetermined. The experiments are designed according to methods routinelypracticed in the art with respect to choice of an ALL cell line, numberof animals per group, dosing and administration schedule of the testgroups and controls, and statistical analytical methods. For example,ALL Nalm-6 cells are tagged with green fluorescent protein (GFP) or DiD(a carbocyanine fluorescent dye) and then engrafted into mice (1×10⁶cells per mouse). Approximately one week after administration of thetagged cells to the animals, groups of mice (6 per group) are treated asfollows. Group 1 (Control) receives vehicle (PBS) only. Each animal inGroup 2 receives an E-selectin antagonist (e.g., Compound 25 (40 mg/kg))daily on days 1, 2, and 3. The animals in Group 3 receive achemotherapeutic drug (e.g., doxorubicin (DOX) (2 mg/kg)) daily on days1, 2, and 3 (called Dose 1). The mice in Group 4 each receive Compound25 (40 mg/kg) three hours prior to DOX administration (2 mg/kg) once aday on days 1, 2, and 3. Group 5 receives DOX at a dose of 3 mg/kg dailyon days 1, 2, and 3 (called Dose 2). In Group 6, each animal receivesCompound 25 (40 mg/kg) three hours prior to DOX administration (3 mg/kg)once a day on days 1, 2, and 3. The mice are observed for up to twomonths. Survival, circulating leukemic cells, and leukemic burden in thebone marrow are determined during the observation period. The number ofcirculating leukemic cells is determined by in vivo flow cytometry.Intravital microscopy is performed to determine leukemic burden in thebone marrow.

Example 5 Effects of Treatment With an E-Selectin-Specific Antagonist ina Pancreatic Cancer Animal Model

The effectiveness of an E-selectin antagonist in a pancreatic canceranimal model is determined. The pancreas of male athymic nu/nu mice (4-6weeks old) are injected orthotopically with S2.013 pancreatic cancercells. Six groups of animals (e.g., 15 mice per group) receive thefollowing treatments beginning approximately 7 days after injection ofthe pancreatic cancer cells, Alternatively, the animals receivetreatments when small tumors are perceptible. Group 1 (Control) receivesvehicle (PBS) only. Group 2 receives gemcitabine twice weekly (2/week)for four weeks. Group 3 receives an E-selectin antagonist (e.g.,Compound 25 (40 mg/kg)) twice daily (BID) for four weeks. Group 4receives Compound 25 (40 mg/kg) once daily (qD) for four weeks. Group 5receives gemcitabine in combination with Compound 25 BID. Group 6receives gemcitibine (dosed twice daily for four weeks) in combinationwith Compound 25, which is administered once daily. Tumor burden isdetermined by ultrasound or 2-deoxyglucose (2DG) imaging. The animalsare sacrificed approximately 4 weeks after treatment.

Example 6 Effects of Treatment With an E-Selectin-Specific Antagonist(Compound 25) in an Animal Model of Venous Thromboembolism (VTE) AnimalModel

Most animal models of venous thromboembolism do not test compounds undercontinuous blood flow, but rather induce thrombosis through ligation orballoon catheterization. A more clinically relevant model was developedin which injury is transiently induced in the presence of continuousblood flow and exposure to normal blood levels of circulating testcompound (see, e.g., Diaz et al., Thromb. Haemot. 104: 366-375 (2010)).A microelectrode is implanted in the inferior vena cava and a current of250 uAmps is applied for 15 minutes. Typical endothelial dysfunctionfound in venous disease was demonstrated by electron microscopy,immunohistochemistry, inflammatory cell counts, and biomarkers ofthrombosis. Ultrasound imaging further detected the formation ofthrombus in real time under blood flow (see, e.g., Diaz et al., supra).

Male C57BL/6J mice underwent an electrolytic inferior vena cava (IVC)model to produce a non-occlusive thrombosis via electrical stimulation(250 μAmp). Animals were divided into prophylactic or treatment groups.Both groups included the following: non-thrombosed animals (TC, nosurgery or drug), 2 Day sham (needle inside the IVC and no current ordrug), 2 Day CTR (No Treatment: current and no drug), 2 Day Compound 25(10 mg/kg IP BID), and low molecular weight heparin (LMWH) (LOVENOX®, 6mg/kg subcutaneously once a day). Animals were divided into prophylacticor treatment groups. Mice in the prophylactic group were dosed one daypre-thrombus induction through day 1. Animals in the treatment groupsreceived the first dose of the drug following thrombus induction onday 1. Mice were euthanized 2 days post-thrombosis for tissue harvestand blood collection for the following evaluations: thrombus weight;vein wall inflammatory cell counts per high power field; veinwall-thrombus histology; and intra-thrombus polymorphonuclear cell (PMN)counts. A separate group of mice received IV administration of compoundsfor tail bleeding time evaluation (seconds).

Thrombus was induced in the inferior vena cava of mice as describedabove in the electrolytic inferior vena cava model (EIM). After injury,the E-selectin specific antagonist, Compound 25 (Example 1), wasadministered as a treatment twice a day at 10 mg/kg. Another cohort ofmice received low molecular weight heparin (LMW heparin) (Lovenox, oncea day, 6 mg/ml). As noted above, on the second day after thrombusinduction, the inferior vena cava was removed and weighed. No electrodeswere implanted in Control mice. Electrodes were implanted but noamperage was delivered in the inferior vena cava of mice in the Shamcohort. As shown in FIG. 4, treatment with Compound 25 at 10 mg/kg afterinjury significantly decreased venous thrombus formation (Compound 25vs. No treatment, P=0.0271) as did LMW heparin (LMW heparin vs. Notreatment, P=0.0203). All mice pre-treated prophylactically withCompound 25 or LMWH followed the same pattern of decreasing thrombusweight 2 days post injury (P<0.05).

Example 7 Effect of Compound 25 on Time Required to Form a Clot

To compare anti-coagulant properties of LMW heparin (LMWH) and Compound25 (see Example 1), bleeding times in mice were evaluated. Testcompounds were injected via the penile vein in mice and after 5 minutesthe tail vein was nicked with a scalpel. The tail was then placed in atube of isotonic saline and the time necessary to blot the wound asrecorded.

LMW heparin is a known anti-coagulant. As shown in FIG. 5, LMW heparindelayed clotting over 4 times longer than control bleeding times,whereas Compound 25 slightly delayed clotting. LMWH at 6 mg/kg dosesignificantly elevated tail bleeding times in mice versus controls(341±27, 491±60 vs. 82±6 seconds, P<0.01). Compound 25 (10 mg/kg, IV)had significantly lower tail bleeding times compared to an IV dose ofLMWH (6 mg/kg, P<0.01). Compound 25 is a significant improvement inreducing bleeding time over LMW heparin.

Vein Wall Morphometries and Histology

Mice that were treated with Compound 25 after injury had significantlydecreased vein wall monocyte extravasation compared to controls(P<0.05). When animals were treated with Compound 25 or LMWHprophylactically (i.e., prior to injury), significantly decreased veinwall PMN extravasation was observed 2 days post thrombosis (P=0.027 andP=0.007 respectively). The same pattern held true for prophylaxis withCompound 25 and LMWH on vein wall monocyte extravasation at the sametime point (P<0.01).

Intra-Thrombus PMN Counts

Compound 25 administered as a prophylactic therapy significantlydecreased intra-thrombus cell counts versus control animal (14.5±3.7 vs.37.4±4.7 PMNs/HPF, P=0.009), and these animals had decreased venousthrombus burden. Only mice that received Compound 25 therapy visuallyhad more intra-thrombus vascular channels compared to control animalsand mice receiving LMWH therapy.

The various embodiments described above can be combined to providefurther embodiments. All U.S. patents, U.S. patent applicationpublications, U.S. patent applications, non-U.S. patents, non-U.S.patent applications, and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary, to employ concepts of thevarious patents, applications, and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1-52. (canceled)
 53. A compound having the formula (I):

or a pharmaceutically acceptable salt, isomer, tautomer, hydrate orsolvate thereof, wherein: R¹ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈alkynyl, C₁-C₈ haloalkyl, C₂-C₈ haloalkenyl or C₂-C₈ haloalkynyl; R² isa linker-non-glycomimetic moiety, wherein the linker is—C(═O)NH(CH₂)₁₋₄NHC(═O)— and the non-glycomimetic moiety comprisespolyethylene glycol; R³ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,C₁-C₈ haloalkyl, C₂-C₈ haloalkenyl, C₂-C₈ haloalkynyl or cyclopropyl; R⁴is —OH or —NZ¹Z² where Z¹ and Z² are each independently H, C₁-C₈ alkyl,C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl, C₂-C₈ haloalkenyl orC₂-C₈ haloalkynyl or wherein Z¹ and Z² join to form a ring; R⁵ is C₃-C₈cycloalkyl; R⁶ is —OH, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈haloalkyl, C₂-C₈ haloalkenyl or C₂-C₈ haloalkynyl; R⁷ is —CH₂OH, C₁-C₈alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₁-C₈ haloalkyl, C₂-C₈ haloalkenylor C₂-C₈ haloalkynyl; and R⁸ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈alkynyl, C₁-C₈ haloalkyl, C₂-C₈ haloalkenyl or C₂-C₈ haloalkynyl. 54.The compound of claim 53, wherein the linker is —C(═O)NH(CH₂)₂NHC(═O)—.55. The compound of claim 53, wherein the non-glycomimetic moietycomprises 1 to 100 ethylene oxide units.
 56. The compound of claim 53,wherein the linker is —C(═O)NH(CH₂)₂NHC(═O)— and the non-glycomimeticmoiety comprises 1 to 100 ethylene oxide units.
 57. The compound ofclaim 56, wherein the non-glycomimetic moiety comprises 2 to 100, 2 to50, 2 to 20, or 2 to 15 ethylene oxide units.
 58. The compound of claim56, wherein the non-glycomimetic moiety comprises 5 to 100, 5 to 40, or5 to 20 ethylene oxide units.
 59. The compound of claim 56, wherein thenon-glycomimetic moiety comprises 2 to 20 ethylene oxide units.
 60. Thecompound of claim 56, wherein the non-glycomimetic moiety comprises 2 to15 ethylene oxide units.
 61. The compound of claim 56, wherein thenon-glycomimetic moiety comprises 5 to 20 ethylene oxide units.
 62. Apharmaceutical composition comprising the compound of claim 53 andoptionally at least one pharmaceutically acceptable excipient.
 63. Amethod for treating, decreasing, inhibiting, or reducing the likelihoodof metastasis of cancer cells in a subject, comprising administering tothe subject the pharmaceutical composition of claim
 62. 64. A method forinhibiting infiltration of cancer cells into bone marrow in a subject,said method comprising administering to the subject the pharmaceuticalcomposition of claim
 62. 65. A method for inhibiting adhesion of a tumorcell that expresses a ligand of E-selectin to an endothelial cellexpressing E-selectin, said method comprising contacting the endothelialcell with a pharmaceutical composition comprising (a) a pharmaceuticallyacceptable excipient and (b) the compound of claim 53 permitting thecompound to interact with E-selectin present on the endothelial cell,thereby inhibiting binding of the tumor cell to the endothelial cell.66. The method of claim 65, wherein the endothelial cell is present inthe bone marrow.
 67. A method for treating a cancer in a subjectcomprising administering to the subject (a) the pharmaceuticalcomposition of claim 62 and (b) at least one of (i) chemotherapy and(ii) radiotherapy.
 68. A method for treating, decreasing, inhibiting, orreducing the likelihood of thrombosis in a subject, comprisingadministering to the subject the pharmaceutical composition of claim 62.69. A method for enhancing hematopoietic stem cell survival in asubject, comprising administering to the subject the pharmaceuticalcomposition of claim
 62. 70. The method of claim 67, wherein the subjecthas received or will receive chemotherapy or radiotherapy or bothchemotherapy and radiotherapy.